Expression of multiple myeloma associated markers in bone marrow spicules using a novel immunohistochemical technique.

Immunohistochemistry (IHC) is an important tool used for diagnosis and prognosis of several hematological malignancies, and it frequently is used for quantitative and qualitative analysis of expression of different protein biomarkers in tissue sections. To understand the histopathological alterations in multiple myeloma (MM), IHC analysis of bone marrow (BM) biopsy is commonly used. Owing to the harsh decalcification process generally used for processing of bone marrow biopsies, however, protein epitopes occasionally are rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin clot matrix that allows IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Using paired BM spicule-clot and BM core biopsies from patients diagnosed with multiple myeloma, we studied six MM related antibodies including kappa and lambda immunoglobulin light chains, CD56, CD138, CYR61 and DKK1.

Identification of Legionella spp. by 19 European reference laboratories: results of the European Working Group for Legionella Infections External Quality Assessment Scheme using DNA sequencing of the macrophage infectivity potentiator gene and dedicated online tools.

Identification of Legionella spp. can be achieved by DNA sequencing of the macrophage infectivity potentiator (mip) gene. The External Quality Assurance (EQA) scheme described in this report is the first to assess the proficiency of laboratories using this methodology. The results obtained from two EQA distributions sent to European reference laboratories involved in Legionella outbreak control and environmental monitoring are presented. Each distribution contained a panel of ten coded Legionella strains. All strains were from clinical and environmental sources and were considered to be wild-type strains. Participants used dedicated online tools to compare sequence text files against a database of known Legionella spp. The majority of centres (seven of ten, and 11 of 12) correctly identified all strains tested, in the first and second distributions, respectively. Typically, sequence similarity values of 98-100% were obtained when the test strains were compared with sequences contained in the database. In all but one case, lower values indicated a poor quality sequence. The exception was associated with the identification of a putative new species in the first panel. Genotypic identification of Legionella can be achieved by the use of standard protocols, dedicated identification libraries, and online tools. EQA schemes provide an independent measure of performance, and it is recommended that laboratories performing these techniques participate in such schemes, thereby allowing optimisation of and improvements in their performance.

Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. The importance of angiogenic factors such as VEGF, while clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Human hematopoietic tumor cell lines, representing multiple lineages and diseases, produce and secrete VEGF and express at least one of its two receptors. Exposure of human vascular endothelial cells to VEGF increased the expression of several hematopoietic growth factors known to be involved in myeloma including interleukin-6 (IL-6). Bone marrow samples from patients diagnosed with multiple myeloma were examined for expression of VEGF and its receptors. VEGF protein production was detected in malignant plasma cells from 78% of the myeloma patients studied. While expression of the Flt-1 and KDR receptors was not observed in the malignant plasma cells, both were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor. In bone marrow clot sections from normal allogeneic donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myelocytes, macrophages, and megakaryocytes. In vitro colony-forming assays using patient-derived material revealed that antibody neutralization of VEGF resulted in an inhibition of colony growth, whereas the addition of recombinant human VEGF stimulated colony formation. Neutralization of VEGF activity also suppressed the generation of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from bone marrow mononuclear cells. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through paracrine and perhaps autocrine mechanisms.

Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.

Cocaine injection and coxsackievirus B3 infection increase heart disease during murine AIDS.

Coxsackievirus initiates myocarditis especially in the immunologically deficient or immature. To test whether Coxsackievirus B3 (CVB3) induced pronounced cardiomyopathy during severe immune dysfunction of murine AIDS, female C57BL/6 mice were infected with LP-BM5 retrovirus and superinfected with CVB3. Some were also injected daily with cocaine hydrochloride in 0.9% saline solution (30 mg/kg) intraperitoneally, because cocaine also suppresses cellular immune response. Heart tissue was analyzed histopathologically. Mice experiencing concurrent retrovirus and Coxsackievirus infection had a high degree of cardiac lesions consistent with myopathy compared with findings in uninfected animals (p <.05). Cocaine injection during murine retrovirus infection greatly exacerbated the pathogenesis of Coxsackievirus infection. C57BL/6 mice, essentially resistant to Coxsackievirus-induced cardiomyopathy, became susceptible during the immune dysfunction in murine AIDS. This suggests that retrovirus infection causes conditions favoring Coxsackie-induced cardiac lesions. Interleukin (IL)-2 and IL-4 expression by splenocytes from the dually infected retrovirus and Coxsackievirus group showed no significant differences when the animals were also cocaine treated. However tumor necrosis factor TNF-alpha production was significantly decreased in dually infected retrovirus + Coxsackievirus mice treated with cocaine, compared with findings in various controls (p <.05).

Thioredoxin, a putative oncogene product, is overexpressed in gastric carcinoma and associated with increased proliferation and increased cell survival.

Human thioredoxin is a putative oncogene that may confer both a growth and survival advantage to tumor cells. Overexpressed thioredoxin mRNA has been found in both primary human lung and colorectal cancers. To determine the intratumor distribution and amount of thioredoxin protein in human primary carcinomas, we developed an immunohistochemical assay for thioredoxin in paraffin-embedded tissue. We then studied 10 patients with primary high-risk gastric carcinoma. To further relate thioredoxin protein overexpression to cell death and survival, we used a paraffin-based in situ end-labeling (ISEL) assay. To delineate proliferation, we used the nuclear proliferation antigen detected by Ki-67. In this survey, we found that thioredoxin was localized to tumor cells and overexpressed compared with normal gastric mucosa in 8 of 10 gastric carcinomas. The thioredoxin was found at high levels in 5 of the 8 overexpressing carcinomas. The overexpression of thioredoxin was typically found in both a nuclear and cytoplasmic location in the neoplastic cells. There was a significant positive correlation (P = .0061) with cancer cell proliferation measured by Ki-67. There was a significant negative correlation (P = .0001) with DNA damage measured by the ISEL assay, suggesting decreased apoptosis and increased carcinoma cell survival. Thus, human primary gastric tumors that are highly expressive of thioredoxin have both a higher proliferative rate and a higher survival rate than tumors that do not express thioredoxin. With these newly developed assays in hand, it is now feasible to question whether this thioredoxin-related combined growth and survival advantage translates into poor clinical outcome.

Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.

pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

Correlation of cytotoxicity and protein-associated DNA strand breaks for 2-(arylmethylamino)-1,3-propanediols.

A mechanism of action study was performed with 14 novel DNA binding agents characterized structurally as 2-(arylmethylamino)-1,3-propanediols (AMAPs). Correlations between 8226 myeloma cell colony formation and DNA damage were performed using soft agar colony-forming assays and alkaline elution filter techniques respectively. The frequency of double-stranded breaks (DSBs), single-stranded breaks (SSBs) and DNA-protein cross-links were compared with cell growth inhibitory potency. Highly potent AMAPs in the colony formation assays included 91U86, an N-methyl-5-benzo(c)carbazole derivative, 773U82, a 3-substituted fluoranthene derivative, and crisnatol (770U82), the 6-substituted chrysene derivative. There was a high frequency of SSBs and DSBs with many analogues, but only SSBs occurred in a concentration-dependent fashion. Using regression analysis, the degree of single-strand damage correlated with cytotoxic potency for the AMAPs, with an R-value of 0.57 (P = 0.04). By gel electrophoresis assays, three clinically tested AMAPs, crisnatol BW 770U82, BW 502U83 and BW 773U82, were shown to inhibit the decatenation of pBR 322 DNA by purified topoisomerase-II (TOPO-II) enzymes. These results suggest that while some active AMAPs, such as crisnatol (BW 770U82), BW 502U83 and BW 773U82, inhibit TOPO-II enzymes, leading to protein-associated SSBs, other mechanisms, which do not involve DNA strand damage, must also contribute to the cytotoxic effects of this class of antitumor compounds. Intercalation has been well documented for these drugs and this may explain some of the growth inhibitory activity of the AMAPs.

Evidence for cytoplasmic P-glycoprotein location associated with increased multidrug resistance and resistance to chemosensitizers.

A new human myeloma cell line, 8226/MDR10V, was selected from a P-glycoprotein-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox40. Chemosensitizers are also less effective in reversing resistance in the MDR10V compared to the Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox40; however, total cellular P-glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR10V cells as compared to Dox40 cells. The cytoplasmic location of P-glycoprotein in the MDR10V cells is associated with a redistribution of doxorubicin. In Dox40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR10V cell line compared to the Dox40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR10V cells compared to Dox40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR10V cells compared to Dox40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR10V cells compared to Dox40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.

Development of an orthotopic SCID mouse-human tumor xenograft model displaying the multidrug-resistant phenotype.

Multiple myeloma is a plasma cell malignancy which is generally incurable in spite of a high initial response to chemotherapy. While animal models of myeloma are known, the recent developments of human xenografts in nude and SCID mice suggests a promising experimental model. The SCID model, in particular, holds promise because these animals readily accept hematopoietic and lymphoid transplantation and do not generally develop graft versus host reaction. We have developed two drug-resistant variants of the human multiple myeloma cell line ARH-77 by in vitro exposure to gradually increasing concentrations of doxorubicin (ARH-D60) or mitoxantrone (ARM-80). When injected into irradiated SCID mice, the ARH-D60 cell line grew in an orthotopic pattern with the development of osteolytic lesions. This is in contrast to the 8226/C1N human myeloma cell line which grows in a disseminated but nonorthotopic manner in the SCID mouse. Both the ARH-D60 and ARM-80 cell lines are resistant to doxorubicin and cross-resistant to mitoxantrone, vinca alkaloids, taxol and m-AMSA while maintaining sensitivity to antimetabolites and alkylating agents. Growth characteristics and cell cycle kinetics, including S-phase, were not altered in the resistant sublines. The ARH-D60 and ARM-80 cell lines both displayed a classic multidrug-resistance (MDR) phenotype which was partially reversed by the addition of verapamil. These two cell lines represent the first MDR human myeloma cell lines which have demonstrated an orthotopic growth pattern in the SCID mouse and thus may be of value in studying the pathophysiology of this disease.

P-glycoproteins and multidrug resistance.

Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases. Studies have demonstrated that this form of drug resistance occurs both in cultured tumor cell lines as well as in human cancers. P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural product drugs from the cell before they have a chance to exert their cytotoxic effects. Expression of the MDR1 gene product has been associated with a poor prognosis in clinical studies. It has been demonstrated in the laboratory that resistance mediated by the P-glycoprotein may be modulated by a wide variety of compounds. These compounds, which include verapamil and cyclosporin, generally have little or no effect by themselves on the tumor cells, but when used in conjunction with antineoplastic agents, they decrease, and in some instances eliminate, drug resistance. Clinical trials to modulate P-glycoprotein activity are underway at the present time to determine if such strategies will be feasible. Although the P-glycoprotein is expressed in many cell lines and occurs in patient tumors, its expression is not a universal feature of multidrug resistance, suggesting that other mechanisms are operating.

Cardiotoxicity in the SCID mouse following administration of doxorubicin and cyclosporin A.

Multiple myeloma is a plasma cell malignancy which is generally incurable in spite of a high initial response to chemotherapy. Relapsing disease commonly heralds an increase in the incidence of drug resistance which is often mediated by the product of the MDR-1 gene, P-glycoprotein (Pgp). One approach to modulating drug resistance due to Pgp overexpression has involved the use of agents known as chemomodulators which inhibit its function. We have developed a human xenograft model of multiple myeloma using the SCID mouse to evaluate the efficacy and toxicities of new MDR-1 chemomodulators. Cyclosporin A (CsA) is a widely used immunosuppressant which has been demonstrated to be a potent inhibitor of Pgp in vitro at concentrations which are clinically achievable. Preliminary studies revealed an acute toxicity in our SCID model which was associated with the combination of CsA and doxorubicin, and which was not observed with either drug alone, nor with cremaphor, the vehicle for CsA. In the current study, non-tumor bearing SCID mice were dosed with doxorubicin or the combination of doxorubicin with cremaphor, verapamil or CsA. Animals were sacrificed and tissues harvested for morphologic examination and for HPLC analysis of doxorubicin levels. In all tissues examined, there was a marked increase in tissue levels of doxorubicin when combined with CsA. Results also revealed a higher incidence and severity of myocardial damage in those animals receiving the combination of doxorubicin and CsA than in those receiving other combinations. The elevations in tissue levels observed with doxorubicin and CsA may contribute to the acute toxicities observed in the SCID mouse model.

Severe combined immunodeficiency (SCID) mouse modeling of P-glycoprotein chemosensitization in multidrug-resistant human myeloma xenografts.

We have established a reproducible in vivo model of human multiple myeloma in the severe combined immunodeficiency (SCID) mouse using both the drug-sensitive 8226/S human myeloma cell line and the P-glycoprotein-expressing multidrug-resistant 8226/C1N subline. As demonstrated previously, the SCID mouse is well suited as a model for myeloma because: (a) human SCID xenografts are readily attained; (b) human myeloma xenografts are readily detected by their immunoglobulin secretion; and (c) differential therapy effects in drug-sensitive versus drug-resistant cell lines are readily demonstrable by monitoring mouse urinary human immunoglobulin output. In the current study, we have utilized this model to evaluate the in vivo efficacy of chemomodulators of P-glycoprotein-related multidrug resistance. In our initial experiments, doxorubicin alone was effective in treating the 8226/S human myeloma xenografts but had no effect on the drug-resistant 8226/C1N xenografts, in the absence of the chemosensitizing agent verapamil. In subsequent experiments, the combination of verapamil and doxorubicin resulted in both a decrease in human lambda light chain urinary excretion and an increase in survival of those animals bearing the 8226/C1N tumor. The median survival time of animals injected with 8226/C1N cells and subsequently treated with doxorubicin was 48.6 +/- 7 days, which compared to a survival of 89.6 +/- 18 days in animals receiving the 8226/S cell line and treated with doxorubicin alone (P < 0.001). When verapamil was added to the treatment regimen of those animals bearing the 8226/C1N xenografts, there was a 179% increase in their life span (P < 0.001), which corresponded with the observed decreased light chain in the urine. In animals receiving multiple courses of chemotherapy, an attenuated response to verapamil and doxorubicin was observed, in a manner analogous to the clinical setting of human drug-resistant myeloma escape from chemosensitivity. The SCID human myeloma xenograft model thus offers a means of evaluating the in vivo efficacy and potential toxicities of new therapeutic approaches directed against P-glycoprotein in multidrug-resistant human myeloma.

A review: the active peptide of lactoferrin.

A potent antimicrobial peptide, 'lactoferricin', was found to be generated upon gastric pepsin cleavage of lactoferrin. The active peptide consists mainly of a loop of 18 amino acid residues, derived from the N-terminal region of the lactoferrin molecule. Like various other antimicrobial peptides that display membrane-disruptive properties, it contains a high proportion of basic amino acid residues. A physiologically diverse range of micro-organisms was tested and found to be susceptible to inhibition by this natural peptide including Gram-negative and Gram-positive bacteria, yeasts and filamentous fungi. Its antimicrobial effect against sensitive micro-organisms was lethal. Electron microscopy studies revealed that it induces a profound change in cell ultrastructural features and causes substantial cell damage in bacteria and fungi. These findings suggest the possibility that active peptides of lactoferrin may have a role in the host defense against microbial disease. If produced in substantial quantities in vivo such peptides could have important physiological significance, especially in nursing infants.

Effect of lactoferricin B, a pepsin-generated peptide of bovine lactoferrin, on Escherichia coli HB101 (pRI203) entry into HeLa cells.

Bovine lactoferrin and a pepsin-generated peptide of bovine lactoferrin, known as lactoferricin B, were tested for an ability to influence the cell-invasive properties of an Escherichia coli HB101 strain carrying the plasmid pRI203, which encodes the Yersinia pseudotuberculosis inv gene. At non-cytotoxic and non-bactericidal concentration (0.5 mg/ml) lactoferricin B lowered by about tenfold the cell invasion capability of E. coli HB101 (pRI203), whereas no effect was observed when bovine lactoferrin was added during the infection of HeLa cell monolayers. The step of the invasion process affected by lactoferrin B was the internalization since the adhesion of bacteria to HeLa cells was unaltered in the presence of the peptide. Latex beads coated with lactoferrin B bound to HeLa cell monolayers and induced the agglutination of bacterial cells, indicating that this highly cationic peptide interacts directly with both eukaryotic and bacterial surfaces. Moreover, we demonstrated that the anti-invasive effect induced by lactoferrin B was reversed when the medium was supplemented with Ca2+, Mg2+ and Fe2+ ions which diminished its affinity binding. Our findings suggest that lactoferrin B effectiveness towards E. coli HB101 (pRI203) invasion is correlated to its binding capability on the eukaryotic and bacterial cell surfaces.