Nammalwar Sriranganathan

Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System

BACKGROUND There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Biological properties of RB51; a stable rough strain of Brucella abortus.

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.

Anti‐inflammatory Properties of Cerium Oxide Nanoparticles

The valence and oxygen defect properties of cerium oxide nanoparticles (nanoceria) suggest that they may act as auto-regenerative free radical scavengers. Overproduction of the free radical nitric oxide (NO) by the enzyme inducible nitric oxide synthase (iNOS) has been implicated as a critical mediator of inflammation. NO is correlated with disease activity and contributes to tissue destruction. The ability of nanoceria to scavenge free radicals, or reactive oxygen species (ROS), and inhibit inflammatory mediator production in J774A.1 murine macrophages is investigated. Cells internalize nanoceria, the treatment is nontoxic, and oxidative stress and pro-inflammatory iNOS protein expression are abated with stimulation. In vivo studies show nanoceria deposition in mouse tissues with no pathogenicity. Taken together, it is suggested that cerium oxide nanoparticles are well tolerated in mice and are incorporated into cellular tissues. Furthermore, nanoceria may have the potential to reduce ROS production in states of inflammation and therefore serve as a novel therapy for chronic inflammation.

Brucellosis: a re-emerging zoonosis.

Brucellosis, especially caused by Brucella melitensis, remains one of the most common zoonotic diseases worldwide with more than 500,000 human cases reported annually. The bacterial pathogen is classified by the CDC as a category (B) pathogen that has potential for development as a bio-weapon. Brucella spp. are considered as the most common laboratory-acquired pathogens. The geographical distribution of brucellosis is constantly changing with new foci emerging or re-emerging. The disease occurs worldwide in both animals and humans, except in those countries where bovine brucellosis has been eradicated. The worldwide economic losses due to brucellosis are extensive not only in animal production but also in human health. Although a number of successful vaccines are being used for immunization of animals, no satisfactory vaccine against human brucellosis is available. When the incidence of brucellosis is controlled in the animal reservoirs, there is a corresponding and significant decline in the incidence in humans.

Brucellosis vaccines: past, present and future

The first effective Brucella vaccine was based on live Brucella abortus strain 19, a laboratory-derived strain attenuated by an unknown process during subculture. This induces reasonable protection against B. abortus, but at the expense of persistent serological responses. A similar problem occurs with the B. melitensis Rev.1 strain that is still the most effective vaccine against caprine and ovine brucellosis. Vaccines based on killed cells of virulent strains administered with adjuvant induced significant protection but also unacceptable levels of antibodies interfering with diagnostic tests. Attempts were made to circumvent this problem by using a live rough strain B. abortus 45/20, but this reverted to virulence in vivo. Use of killed cells of this strain in adjuvant met with moderate success but batch to batch variation in reactogenicity and agglutinogenicity limited application. This problem has been overcome by the development of the rifampicin-resistant mutant B. abortus RB51 strain. This strain has proved safe and effective in the field against bovine brucellosis and exhibits negligible interference with diagnostic serology. Attempts are being made to develop defined rough mutant vaccine strains that would be more effective against B. melitensis and B. suis. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens, with or without adjuvants. Limited success has been obtained with these or with DNA vaccines encoding known protective antigens in experimental models and further work is indicated.

Flagellin Is an Effective Adjuvant for Immunization against Lethal Respiratory Challenge with Yersinia pestis

ABSTRACT Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response.

Suppression of Salmonella growth by wild-type and large-plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters.

The bacteriophage Felix O1, a member of Myoviridae, is specific for, and possesses a broad host range within, the genus Salmonella. This work explores a Felix O1 phage-based intervention for Salmonella enterica serotype Typhimurium DT104 that is potentially applicable at several stages of animal production and processing. A variant of Felix O1 was obtained that produces a larger, clearer plaque phenotype (LP) on Salmonella Typhi than wild-type Felix O1 (WT) does, not unlike r mutants of phage T4. LP exhibited slightly more extensive overall suppression of Salmonella Typhi in brain heart infusion (BHI) broth, as ascertained on the basis of culture turbidity (optical density at 600 nm). Both phage variants suppressed log phase BHI broth cultures containing 8.2 x 10(6) CFU of Salmonella Typhimurium DT104 per ml. A PFU/CFU ratio of 1.0 was effective for WT and LP, whereas increasing the PFU/CFU ratio to 5.0 did not increase suppression. Untreated Salmonella-contaminated frankfurters were compared with treated samples (PFU/CFU ratio, 1.9 x 10(4)) to test WT and LP for their ability to suppress Salmonella growth on chicken frankfurters contaminated with 300 CFU of Salmonella Typhimurium DT104. Suppression levels of 1.8 and 2.1 log units were achieved with WT and LP, respectively (P = 0.0001), but no difference was found between the performances of the two variants (P = 0.5088).

Flagellin-F1-V Fusion Protein Is an Effective Plague Vaccine in Mice and Two Species of Nonhuman Primates

ABSTRACT A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4°C and at least 168 days at 25°C. At between 29 and 84 days at 37°C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.

A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies.

Alamar Blue in the microenvironment of activated cells, undergoes color change and also becomes fluorescent. By using the Alamar Blue dye, we have reported a non-radioactive colorimetric assay to indirectly determine proliferation of murine lymphocytes. We further show that the pattern of mitogen-induced proliferation assessed fluorometrically was comparable to the 3H-thymidine incorporation assay (3H-Tdr assay). Of practical importance is that the color/fluorescence changes were stable at 4 degrees C in the dark for 3-4 weeks. In immunological studies, it is important to further analyze lymphocytes that have undergone activation and/or proliferation. This is not possible with the standard 3H-Tdr assay, which requires lysis of cells. In contrast, the Alamar Blue-based non-radioactive assay does not require cell lysis. We therefore tested the hypothesis that further analysis of lymphocytes is possible, after assessing the proliferation using Alamar Blue. Following assessment of proliferation in a 72-h culture, the Alamar Blue dye was washed-off and cells were re-utilized to perform additional immunological analysis. Short-term exposure of lymphocytes to Alamar Blue was not detrimental to lymphocytes, as assessed by trypan blue exclusion and the propidium iodide (PI) assays. Exposure of dexamethasone-treated cells to Alamar Blue did not interfere with the performance of apoptosis assays, such as flow cytometric analysis of PI-stained cells and microscopic examination of ethidium bromide/acridine orange-stained cells. In addition, prior exposure of lymphocytes to Alamar Blue did not affect the analysis of chromosomal aberrations or the visualization of cell surface antigens by flow cytometry. Further, the expression of cytokine mRNA in lymphocytes previously exposed to Alamar Blue was similar to unexposed cells. Together, a notable advantage of this assay is that it now enables the investigator to maximize information by following or correlating proliferation with other immunologic events in the same cells.

Overexpression of Protective Antigen as a Novel Approach To Enhance Vaccine Efficacy of Brucella abortus Strain RB51

ABSTRACT Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.