HIV gp120-specific cell-mediated immune responses in mice after oral immunization with recombinant Salmonella

Salmonella is of great interest as a potential human immunodeficiency virus vaccine vector because of its ability to elicit potent mucosal and systemic immune responses when administered orally. To determine whether such a vaccine could elicit an immune response in mice, plasmids expressing HIV gp120-LAI were introduced into attenuated S. typhimurium. Three serial doses of 10(10) recombinant organisms were administered orally to BALB/c mice at 2-week intervals. Immunized mice but not control mice demonstrated proliferative T cell responses to gp120-LAI, comparable in magnitude to the proliferative responses to Salmonella antigens. Immunized mice had detectable serum and intestinal Salmonella-specific IgA and serum Salmonella-specific IgG. However, no gp120-specific antibody was detected in either serum or intestinal washes. These results indicate that live recombinant Salmonella-based vaccine constructs can induce HIV-specific cellular immune responses in vivo.     

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freund's complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.     

Control of immune responses by gene immunization

AIMS OF THE STUDY: To determine the risk factors for local and distant failure in node-negative breast cancer treated with breast-conservative surgery and radiotherapy and to determine the relationship between these two events. MATERIAL AND METHODS: We retrospectively selected 908 patients who received conservative surgery and radiotherapy but no chemotherapy between 1980 and 1995, for a node-negative breast cancer. Patients were divided in two groups according to the status of the margins of resection. All pathology specimens were reviewed. RESULTS: In case of negative margins, the risk factors for local recurrences picked up by the Cox model were histologic multifocality (P = 0.0076), peritumoral vessel invasion (P = 0.021) and age < or = 40 years (P = 0.024), and in case of involved margins, negative oestrogen receptors (P = 0.0012), histologic multifocality (P = 0.0028), and absence of hormonal therapy (P = 0.017). The 10-year local recurrence rate was 18% in case of negative margins and 29% in case of involved margins, although in the latter case patients received high-dose adjuvant radiotherapy. Accordingly, the 10-year distant failure rates were 16% and 27%, respectively. Many arguments suggest that local and distant failures are closely related. CONCLUSION: Patients with histologic multifocality or positive margins are at high risk of local failure and then of distant failure, and require a more aggressive initial treatment.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Receptors on chicken erythrocytes for F42 fimbriae of Escherichia coli isolated from pigs

Haemagglutination of purified F42 fimbriae was found to be inhibited by N-acetyl-galactosamine. Purified F42 fimbrial adhesin reacted with distinct membrane components from chicken erythrocytes (35, 37 and 40 kDa) in immunoblot analysis, suggesting that the binding occurred to proteins or glycoproteins.     

Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.     

Hepatitis C virus envelope DNA-based immunization elicits humoral and cellular immune responses

BACKGROUND: Cholelithiasis affects 10-20% of the USA population, with higher incidence in certain ethnic groups. Obesity is associated with an increase in gallstone formation, reported in up to 45% of morbidly obese patients. Ultrasound is the best diagnostic tool, although its accuracy is less in this particular population. This paper discusses false negative sonographic findings in morbid obesity. METHODS: Retrospective review of 5257 patients submitted to bariatric surgery. Cholecystectomy had previously been performed in 16%. Gallbladder ultrasound was obtained in the remaining group, and cholecystectomy was done based on this information and/or intraoperative observations. Radiology results and surgical findings were correlated with pathology reports. Misread films were reviewed by a radiologist blind to these reports. RESULTS: The series consisted of 88% females. Mean age, weight and percentage overweight were 37 years, 125 kg and 105%, respectively. Cholecystectomy was performed in 3084 patients (59%). Discrepancies between radiological and pathological findings were found in 35 cases (1.1%). Five correct diagnosis of lithiasis also had gallbladder hydrops. Four 'inconclusive' and 20 'negative' studies showed definitive pathology. In six cases of 'non/poor visualization', lithiasis was encountered. CONCLUSIONS: Preoperative gallbladder ultrasound is mandatory in bariatric surgery. Results are accurate and false-negative reports rare if sonographers and radiologists are experienced. Non/poor visualization is usually due to technical problems or gallbladder pathology, not due to the patient's size. False-negative results are commonly caused by soft stones, microlithiasis or polypoid cholesterolosis. Single calculus impacted in the cystic duct can produce hydrops, resulting in a negative sonogram.     

Antiviral immune responses in CTLA4 transgenic mice

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.     

Antibody and cell-mediated immune responses to booster immunization with a new acellular pertussis vaccine in school children

235 healthy 10-12 years old school children were randomly immunized with either a booster dose of diphtheria-tetanus-acellular pertussis (dTap) or diphtheria-tetanus (dT) vaccine. For this booster immunization designed for school children and adults, the quantities of Bordetella pertussis antigens in the dTap vaccine had been reduced to one third of those of the Infanrix vaccine (SmithKline Beecham) commonly used for infants. IgG antibodies and cell-mediated immune (CMI) responses to pertussis toxin (PT), pertactin (PRN) and filamentous hemagglutinin (FHA) were assessed by an enzyme immunosorbent assay and in vitro proliferation of peripheral blood mononuclear cells, respectively. Before immunization, 55%, 80% and 99% of children had detectable serum IgG antibodies to PT, PRN and FHA, whereas CMI response was found in 35%, 27% and 50% of children, respectively. After immunization, a 20-30-fold increase in geometric mean level (GML) of antibodies to the pertussis antigens occurred and CMI response to PT, PRN and FHA was seen in 88%, 94% and 100% of children, respectively. Adverse reactions following the immunization were rare. The results show that booster immunization with an acellular pertussis vaccine with reduced concentrations of antigens induces both antibody and CMI responses and support further studies of this pertussis vaccine in school children.     

Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs

The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1a) livers into PVG (RT1c) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1(1)) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.     

Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans

Numerous experiments performed in humans and animals have revealed that stimulation of mucosal lymphoid inductive sites such as intestinal Peyer's patches results in parallel immune responses manifested by the appearance of S-IgA antibodies in the external secretions of remote glands. However, recent experiments suggest that inductive sites associated with the upper respiratory tract, rectum, and perhaps genital tract may also function as sources of lymphoid cells that populate, with some selectivity, certain remote mucosal effector sites. Furthermore, antigen-specific IgA antibodies can be induced in certain secretions (e.g., female genital tract) not only by immunization in the vicinity of corresponding mucosal tissues (e.g., vagina and rectum) but also by oral and especially intranasal immunization. The ineffectiveness of simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies of strategies for effective delivery systems that would (a) increase the antigen absorption, (b) prevent its degradation, and (c) skew the outcome of immunization to a desired goal (protective response to infectious diseases vs. tolerance; B vs. T cell responses; mucosal vs. systemic). The induction of immune responses at a desired mucosal site can be accentuated with the use of a suitable antigen-delivery system including relevant bacterial or viral vectors, edible transgenic plants expressing microbial antigens, incorporation of antigens in biodegradable microspheres or liposomes, and linkage or coadministration of antigens with cholera toxin B subunit. However, only a few antigen-delivery systems extensively used in animal experimentation have been evaluated for their efficacy in humans. The combination of various immunization routes and the use of suitable antigen-delivery systems may accomplish an important task-the induction of mucosal immune responses at a location relevant to the site of entry of a given pathogen.     

Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.     

Relationship between venous bubbles and hemodynamic responses after decompression in pigs

We present a new pig model for studying relationships between venous gas bubbles and physiologic effects during and after decompression. Sixteen pigs were anesthetized to allow spontaneous breathing. Eight of them underwent a 30-min exposure to 5 bar (500 kPa) followed by a rapid decompression to 1 bar (2 bar/min); the remaining eight served as controls. The pigs were monitored for intravascular bubbles using a transesophageal echocardiographic transducer, and bubble count in the two-dimensional ultrasound image of the pulmonary artery was used as a measure of the number of venous gas bubbles. Effects on physiologic variables of the pulmonary and the systemic circulations were either measured or estimated. We detected venous bubbles in all pigs after decompression, but the interindividual variation was large. The time course of changes in the mean pulmonary artery pressure, in the pulmonary vascular resistance, in the arterial oxygen tension, and in the pulmonary shunt fraction followed the time course of the bubble count. In contrast, such a relationship to the number of venous gas bubbles was not found for the immediate increase in mean arterial pressure and for the changes in the other variables of the systemic circulation. We conclude that the number of venous gas bubbles, as evaluated by the bubble count in the ultrasound image of the pulmonary artery, is clearly related to changes in the variables of the pulmonary circulation in this pig model.     

Chicken egg yolk antibodies against F18ab fimbriae of Escherichia coli inhibit shedding of F18 positive E. coli by experimentally infected pigs

F18ab and F18ac are antigenic variants of a colonizing fimbria commonly found on E. coli associated with postweaning diarrhea and edema disease in pigs. Chicken F18ab antibodies were obtained by immunising hens with purified F18ab fimbriae. For their in vitro characterisation antibodies were isolated from diluted egg yolks by ammonium sulfate precipitation. In vitro adhesion tests demonstrated that the chicken F18ab antibodies inhibited attachment of F18ab positive E. coli bacteria to the intestinal mucosa. Just weaned piglets were experimentally infected with an F18ab positive edema disease strain of E. coli, or with an F18ac positive postweaning diarrhea E. coli strain. The animals were infected on the second day of a period during which chicken F18ab antibodies were added to their feed. During the same period, pigs of the control group received commercial eggs in which no F18 antibodies were detected. In both experimental infections the excretion of the F18 positive strain was reduced in pigs that received the F18ab antibodies as compared to the control animals. The F18ab antibodies diminished the cases of diarrhea and death in animals infected with F18ac positive E. coli.     

Cell-mediated immune responses in patients with paracoccidioidomycosis

The morphology of lymph node tissue from normal hamsters and from hamsters experimentally infected with Treponema pertenue Gauthier was compared by means of light and electron microscopy. The capsules of the lymph nodes from infected hamsters showed an increased thickness in comparison with those of the non-infected animals. The infected lymph nodes differed from normal lymph nodes by small accumulations of neutrophilic leucocytes in the cortical areas. In addition, the amount of intercellular collagenous matrix present between large elongated cells was greatly increased in lymph nodes from infected animals. Electron microscopy of thin sections of infected lymph nodes showed intercellularly located treponemes in the leucocyte infiltration areas. These regions also showed the increased amounts of the collagenous matrix. Treponemes were occasionally found intracellularly in macrophages. These treponemes did not show their typically helical shape, but were present as spherical forms or cysts.     

Antigen-specific T-cell responses in humans after intranasal immunization with a meningococcal serogroup B outer membrane vesicle vaccine

We have studied the ability of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine, when administered intranasally without adjuvant, to induce T-cell responses in humans. A group of 12 vaccinees was immunized with four doses of OMVs (250 micrograms of protein/dose) at weekly intervals, and a single booster dose was given 5 months later. In vitro T-cell proliferation in response to the OMV vaccine, purified PorA (class 1) protein, PorB (class 3) protein, and one unrelated control antigen (Mycobacterium bovis BCG) was measured by [3H]thymidine incorporation into peripheral blood mononuclear cells obtained from the vaccinees before and after the immunizations. The nasal OMV immunizations induced antigen-specific T-cell responses in the majority of the vaccinees when tested against OMVs (7 of 12) and the PorA antigen (11 of 12). None of the vaccinees showed a vaccine-induced T-cell response to the PorB antigen after the initial four doses. Although some individuals responded to all the vaccine antigens after the booster dose, this response was not significant when the vaccinees were analyzed as a group. We have also demonstrated that the PorA antigen-specific T-cell responses correlated with anti-OMV immunoglobulin A (IgA) levels in nasal secretions, with anti-OMV IgG levels in serum, and with serum bactericidal activity. In conclusion, we have shown that it is possible to induce antigen-specific T-cell responses in humans by intranasal administration of a meningococcal OMV vaccine without adjuvant.     

Intestinal immune responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood

An inactivated oral enterotoxigenic Escherichia coli (ETEC) vaccine against ETEC diarrhea was given to 25 adult Swedish volunteers. The vaccine consisted of formalin-killed E. coli bacteria expressing the most common colonization factor antigens (CFAs), i.e., CFA/I, -II, and -IV, and recombinantly produced cholera B subunit (CTB). Immunoglobulin A (IgA) antibody responses in intestinal lavage fluid to CTB and CFAs were determined and compared with corresponding responses in stool extracts and serum as well as with IgA antibody-secreting cell (ASC) responses in peripheral blood. Two doses of vaccine induced significant IgA responses to the different CFAs in lavage fluid in 61 to 87% of the vaccinees and in stool in 38 to 81% of them. The most frequent responses were seen against CFA/I. The magnitudes of the antibody responses against CTB and CFA/I in stool correlated significantly (CTB, P < 0.01; CFA/I, P < 0. 05) with those in intestinal lavage. Intestinal lavage responses against CFAs were best reflected by the ASC responses, with the sensitivity of the ASC assay being 80 to 85%, followed by stool (sensitivity of 50 to 88%) and serum antibody (sensitivity of 7 to 65%) analyses. CTB-specific immune responses were seen in >90% of the vaccinees in all assays.     

Aqueous-based microencapsulation enhances virus-specific humoral immune responses in mice after parenteral inoculation

Vaccines are commonly administered by the parenteral route. Therefore, adjuvant strategies which include parenteral immunization may improve the efficacy of a number of current vaccines. The capacity of aqueous-based microencapsulation to enhance virus-specific IgG responses in mice inoculated intramuscularly with small quantities of antigen was evaluated. Mice were inoculated with either 10(4), 10(3), or 10(2) p.f.u. of microencapsulated rotavirus (bovine strain WC3), placebo microcapsules plus free virus, or virus alone. Mice were subsequently bled 1, 2, 4, 6, and 9 months after inoculation. Microencapsulation of rotavirus enhanced virus-specific humoral immune responses. In addition, virus-containing microcapsules composed of spermine-chondroitin sulfate induced levels of virus-specific antibodies greater than those found after inoculation with virus-containing microcapsules composed of spermine-alginate. Mechanisms by which microencapsulation may enhance virus-specific humoral immunity are discussed.     

Neuroendocrine and immune system responses with spaceflights

Despite the fact that the first human was in space during 1961 and individuals have existed in a microgravity environment for more than a year, there are limited spaceflight data available on the responses of the neuroendocrine and immune systems. Because of mutual interactions between these respective integrative systems, it is inappropriate to assume that the responses of one have no impact on functions of the other. Blood and plasma volume consistently decrease with spaceflight; hence, blood endocrine and immune constituents will be modified by both gravitational and measurement influences. The majority of the in-flight data relates to endocrine responses that influence fluids and electrolytes during the first month in space. Adrenocorticotropin (ACTH), aldosterone, and anti-diuretic hormone (ADH) appear to be elevated with little change in the atrial natriuretic peptides (ANP). Flight results longer than 60 d show increased ADH variability with elevations in angiotensin and cortisol. Although post-flight results are influenced by reentry and recovery events, ACTH and ADH appear to be consistently elevated with variable results being reported for the other hormones. Limited in-flight data on insulin and growth hormone levels suggest they are not elevated to counteract the loss in muscle mass. Post-flight results from short- and long-term flights indicate that thyroxine and insulin are increased while growth hormone exhibits minimal change. In-flight parathyroid hormone (PTH) levels are variable for several weeks after which they remain elevated. Post-flight PTH was increased on missions that lasted either 7 or 237 d, whereas calcitonin concentrations were increased after 1 wk but decreased after longer flights. Leukocytes are elevated in flights of various durations because of an increase in neutrophils. The majority of post-flights data indicates immunoglobulin concentrations are not significantly changed from pre-flight measurements. However, the numbers of T-lymphocytes and natural killer cells are decreased with post-flight conditions. Of the lymphokines, interleukin-2 production, lymphocyte responsiveness, and the activity of natural killer cells are consistently reduced post-flight. Limited head-down tilt (HDT) data suggest it is an effective simulation model for microgravity investigations. Neuroendocrine and pharmacological countermeasures are virtually nonexistent and should become high priority items for future research. Although exercise has the potential to be an effective countermeasure for various neuroendocrine-immune responses in microgravity, this concept must be tested before flights to Mars are scheduled.     

Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune responses and protection from genital challenge

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.