Prophylactic efficacy of high-molecular-weight antigenic fractions of a recent clinical isolate of Leishmania donovani against visceral leishmaniasis

T-cell mediated immune responses are key determinants to the natural course of infection caused by intracellular parasites such as Leishmania. Thus, T-cell activating proteins of these microbes continue to generate active interest particularly in view of their possible role in the design and development of newer and more effective vaccines. We have recently reported the presence of T-cell immunostimulatory antigens with the high-molecular-weight (MW) fractions (134-64.2 kDa) of whole Leishmania donovani antigen (strain 2001), which stimulated variable amounts of IFN-gamma, IL-12 and IL-10 in exposed immune individuals. The present study was undertaken to further evaluate these high-MW antigenic fractions (MW range >100-60 kDa) for potential protective efficacy. The high-MW region of the parasite was resolved into five antigenic fractions (Prep A-E) using continuous elution gel electrophoresis. Prior to in vivo protection studies in hamsters, these fractions were used to evaluate in vitro cellular responses in eight Leishmania-exposed individuals and treated cured hamsters. The protective efficacy of prep (A + B), C, D and E in combination with BCG was evaluated in inbred hamsters using standard immunization protocol. Proliferative responses were seen in all eight of eight exposed individuals to prep D [median stimulation index (SI): 5.2 (range 3.9-7.1)] and E [median SI: 5.6 (range 4.4-8.2)], five of eight individuals to prep B and prep C and three of eight to prep A [median SI: 0.2 (range 0.1-7.2)]. The median proliferative responses to prep D and prep E were significantly higher than to fraction prep A; (P < 0.05) but not to prep B and prep C. However, prep A-E induced equivalent levels of IFN-gamma, IL-10 and IL-12 cytokines. Fractions D and E also exhibited marked parasite inhibition in spleen (52.5% and 73.7%) and liver (65% and 80.2%) as compared with prep (A + B) (23% in spleen and 24% in liver) and prep C (38% in spleen and 24% in liver). Prep D and prep E vaccinated animals showed higher in vitro stimulatory responses (mean SI: 6.6 and 8.8) and nitric oxide (NO) induction (mean NO levels: 6.4 and 10.7 mug/ml) against whole cell extract as compared with other groups. The protection also correlated with presence of suppressed Leishmania-specific IgG levels in prep D and prep E immunized hamsters. These studies indicate the presence of immunostimulatory and protective molecules in 60-80 kDa region of L. donovani, which may be further exploited for developing a subunit vaccine.     

Protective immunity against experimental pulmonary cryptococcosis in T cell-depleted mice

Individuals with defects in T cell-mediated immunity (CMI) are highly susceptible to infection with Cryptococcus neoformans. The purpose of these studies was to determine if protection against experimental pulmonary cryptococcosis can be generated in T cell-deficient hosts. BALB/c mice were depleted of CD4⁺ and/or CD8⁺ T cells or given an isotype control antibody prior to vaccination with a C. neoformans strain, designated H99γ, previously shown to induce protection against C. neoformans infection in immunocompetent mice. Mice depleted of CD4⁺ or CD8⁺ T cells, but not both subsets, survived an acute pulmonary infection with C. neoformans strain H99γ and a subsequent second challenge with wild-type C. neoformans strain H99. We observed a significant increase in the percentage of CD4⁺ and CD8⁺ T cells expressing the activation marker CD69 in the lungs of mice immunized with C. neoformans strain H99γ prior to a secondary challenge with wild-type cryptococci. CD4⁺ T cells within the lungs of immunized mice also appeared to acquire a predominantly activated effector memory cell phenotype (CD69⁺ CD44⁺ CCR7⁻ CD45RB⁻ CD62L⁻) following a second pulmonary challenge with wild-type C. neoformans, compared to CD4⁺ T cells from naïve mice. Lastly, immunization of immunocompetent mice with C. neoformans strain H99γ prior to depletion of CD4⁺ and/or CD8⁺ T cells resulted in significant protection against a second challenge with wild-type C. neoformans. Our studies demonstrate that protective immunity against pulmonary cryptococcosis can be generated in immunosuppressed hosts, thus supporting the development of cryptococcal vaccines.     

Individual antigenic specificity of mouse myeloma proteins

Five murine BALB/c myeloma proteins have been studied for their antigenic specificities: IgG1, IgG2 and three of the IgA class. In each case we could demonstrate individual antigenicity and elicit monospecific antibodies for each protein. Using the passive hemagglutination of sheep red blood cells coated with protein by the monospecific homologous antiserum, and its inhibition by normal immunoglobulins, we tried to determine whether a normal immunoglobulin population of syngeneic mice could exhibit the counterpart of these individual antigenic specificities. The specific counterparts of proteins IgG1 and IgG2 have been found; the quantities of both normal immunoglobulins and of paraproteins needed to ensure the same inhibition of the antiserum were at a ratio of about 1000 : 1. However, we were unable to detect such a counterpart for the three IgA myeloma proteins, although we observed a reaction of antigenic identity between two IgA proteins.     

The antigenic properties of some fractions of Ehrlich's adenocarcinoma of mice

Summary The author studied the antigenic structure of Ehrlich's adenocarcinoma and its globulin fractions in reaction of precipitation in gel. Two tissue antigens were detected in the tumor, both differing from the species antigens; one of these tissue antigens proved to be specific. One tissue antigen was found in the globulin fractions. The latter showed pronounced immunizing activity in experiments with vaccination of mice. Inoculation into the tail with a living tumor was more effective than chemical vaccination with tumor fractions.(Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental' noi Biologii i Meditsiny, Vol. 51, No. 5, pp. 86–91, May, 1961     

Human cell-mediated immune responses to antigenic fractions of Salmonella typhi.

The proliferative responses of peripheral blood mononuclear cells of 10 subjects that had typhoid fever, and healthy volunteers without history of typhoid fever or immunization against disease, were analysed with antigen fractions from two protein extracts of Salmonella typhi. Fractions from each extract were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred to nitrocellulose filters by electroblotting and processed to obtain antigen-bearing nitrocellulose particles for use in lymphocyte cultures. Although the individual proliferative responses were heterogeneous we identified two main immunogenic regions of 29-32 10(3) MW and 45-56 x 10(3) MW for both extracts. Even though there was no one particular antigenic fraction capable of stimulating lymphocytes from all individuals with a previous history of typhoid fever, the combination of three fractions 29-32, 41-45, 63-71 x 10(3) MW could be stimulatory for cells of 90% of these individuals. Also, four subjects that did not respond to unfractionated antigens gave proliferative responses to several fractions of the same extract. We have identified the main immunogenic fractions of S. typhi that might play a role during typhoid infection and postinfection immunity, and merit further purification and characterization.     

Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models

ABSTRACT: BACKGROUND: Trastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models. METHODS: PDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient's ESCC tissues. Similarity between the PDECX models and their corresponding patient's ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation. RESULTS: None of the PDECX models (or their corresponding patient's ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model. CONCLUSIONS: This study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.     

Protective efficacy of mouse serum to the N-propionyl derivative of meningococcal group B polysaccharide

The protective properties of antibodies induced by immunization of mice with a conjugate of tetanus toxoid and the N-propionyl derivative of group B meningococcal polysaccharide (N-Pr-GBMP-TT) have been investigated. Mice immunized with the conjugate produced antibodies which were bactericidal for Neisseria meningitidis strains B:2b:P1.Ham and B:15:P1.16. Passive protection studies indicated that the conjugate serum completely eliminated or reduced considerably levels of bacteremia by the same strains in mice. There was no bactericidal activity or passive protection against a strain of N. meningitidis C:2b:P1.2. Following absorption of the conjugate serum with GBMP the non-absorbed antibody, directed to N-Pr-GBMP, was bactericidal and protected mice against bacteremia with group B meningococci. Thus N-Pr-GBMP antibodies which do not bind to the GBMP are protective in vitro and in vivo.     

Coccidioides immitis fractions which are antigenic for immune T lymphocytes.

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.     

Preclinical pharmacokinetic, biodistribution, and anti-cancer efficacy studies of a docetaxel-carboxymethylcellulose nanoparticle in mouse models

We have developed a polymer conjugate (Cellax) composed of acetylated carboxymethylcellulose (CMC), docetaxel (DTX), and PEG, designed to enhance the pharmacokinetics (PK) and antitumor efficacy of DTX. Our design placed an emphasis on nanoparticle self-assembly to protect DTX during blood transport, stability of the nanoparticle, and PEGylation to enhance PK. Compared to Taxotere, Cellax exhibited a 38.6 times greater area under the curve (AUC), and significantly lower clearance (2.5%) in PK. Less than 10% of DTX was released from Cellax in the blood circulation, indicating that Cellax were stable during blood transport. Cellax reduced non-specific distribution of DTX to the heart, lung and kidney by 48, 90, and 90%, respectively, at 3 h, compared to Taxotere. The uptake of Cellax at 3 h in the liver and spleen was high (15-45 μg DTX/g) but declined rapidly to <10 μg DTX/g in 24 h, and induced no measurable toxicity at 170 mg DTX/kg. Taxotere, on the other hand, displayed non-specific uptake in all the examined normal tissues and induced significant apoptosis in the lung and kidney at 40 mg DTX/kg. The tumor uptake of Cellax was 5.5-fold more than that by Taxotere and the uptake occurred within 3 h after injection and persisted for 10 days. The conjugate exhibited enhanced efficacy in a panel of primary and metastatic mouse tumor models. These results clearly demonstrated that Cellax improved the pharmacokinetics, biodistribution and efficacy of DTX compared to Taxotere with reduced toxicity.     

Protective efficacy of hepatitis B vaccines in neonates

A literature search was carried out to investigate the factors that influence the protective efficacy (PE) of hepatitis B vaccines when given to neonates of hepatitis B surface antigen and e antigen positive mothers. Hepatitis B vaccines with either high or low antigen doses are very effective in preventing chronic hepatitis B infection in neonates at risk, but there is evidence that with lower dosages simultaneous use of hepatitis B immune globulin (HBIG) administration is more important than with higher dosages to elicit good protection (PE ≧ 90%). There is also a tendency for lower dosages to confer high PE less consistently, with noticeably greater numbers of chronic surface antigen carriers in neonates who received a complete vaccination course. Furthermore vaccination courses with higher vaccine dosages give high PEs, without concomitant HBIG administration at birth, provided that the first vaccine dose is given at birth and that the second dose follows within 2 months. © 1994 Wiley-Liss, Inc.     

Identification of phase-specific antigenic fractions of Coxiella burnetti by enzyme-linked immunosorbent assay.

Antigenic fractions of Coxiella burnetii phase variants were identified with an enzyme-linked immunosorbent assay (ELISA). Immune sera from guinea pigs immunized with Formalin-inactivated phase I or phase II whole cells were used to measure the antigenic activity of whole cells and various soluble and particulate preparations. Phase-specific antigens of C. burnetii whole cells and fractions were compared by dose-response curves at different (antigen and antibody) dilutions. Water-soluble extracts prepared by meta-periodate, ether, and phenol extraction of phase I whole cells yielded antigenic fractions which reacted with anti-phase I antibodies. The extraction of phase I whole cells with dimethyl sulfoxide, trichloracetic acid, and Formalin yielded antigenic fractions which detected antibodies in both anti-phase I and -phase II sera. Interestingly, the trichloracetic acid extract of phase I whole cells also contained a component which bound nonimmune immunoglobulin. The sera of animals immunized with whole cells of the phase II Australian QD strain reacted with lipopolysaccharides of the phase I and phase II Nine Mile strains. Therefore, variations in lipopolysaccharide structure among phase variants of C. burnetii were detected as cross-reactions with immune sera from an interspecific strain. Comparisons of immunofluorescence, microagglutination, and the complement fixation assays with the ELISA indicated greater sensitivity and specificity of the ELISA for the measurement of phase-specific antigens and antibodies.     

Lysosomal enzyme changes in mouse spleen cells after antigenic stimulation

Changes in the lysosomal enzyme acid phosphatase have been assayed in mouse spleen cells after injection of the animal with various antigens. Disruption by freezing and thawing or sonication of spleen cells or the large granular fraction showed, 48 hours after injection of antigen, an increase in enzyme level irrespective of the antigen used. In contrast, if non-disrupted lysosomes were examined only cells from animals treated with aggregate-free antigen (BSA) showed an increase in level. Spleen cells from animals treated with the other antigens, heat-aggregated BSA, haemocyanin (KLH) and sheep red cells, showed that the enzyme was less accessible to its substrate, reflected by lower levels of enzyme. The results are discussed in relation to possible changes in the stability of the lysosome membrane and the involvement of lymphocytes.     

Selective binding of chemically defined antigenic peptides to mouse lymphocytes

Immunologically active peptides containing either two different (N? 10? C) or two identical (N? 8? N) antigenic amino acid sequences, bridged by 10 and 8 glycine residues, respectively, were used to study antigen-binding lymphocyte populations in BALB/c mice. Spleen cells from mice immunized with either N? 10? C or N? 8? N were treated with either normal rabbit serum or brain associated anti-thymus (BA Θ) serum and subsequently examined by autoradiography for the numbers of cells binding ¹²⁵I-labeled N? 10? C, and ¹²⁵I-labeled conjugates of either the C antigenic determinant or the N antigenic determinant with poly? D? glutamic acid. In unimmunized mice there were more cells binding the N determinant than the C determinant. After anti-BA Θ treatment, the relative numbers of each population increased, implying that many of these cells were bone marrow-derived. In N? 10? C or N? 8? N immunized mice, the numbers of cells binding the N determinant remained virtually unchanged after anti-BA Θ treatment, indicating an equal distribution of these cells in both bone marrow or thymus-derived populations. However, the C determinant binding population in N? 10? C immunized animals appeared to be mainly of bone marrow origin. Since the C determinant exhibited mainly B cell reactivity, the role of thymus cells in immune responsiveness to a peptide (C? mal? 10? C) containing two C determinants was examined. Only those mice which were reconstituted with both bone marrow and thymus cells after thymectomy and irradiation were capable of responding normally to this antigen, demonstrating thymus involvement even with antigens that appear to be mainly reactive with B cells.     

Cystic fibrosis mouse models

Animal models of cystic fibrosis (CF) are powerful tools that enable the study of the mechanisms and complexities of human disease. Murine models have several intrinsic advantages compared with other animal models, including lower cost, maintenance, and rapid reproduction rate. Mice can be easily genetically manipulated by making transgenic or knockout mice, or by backcrossing to well-defined inbred strains in a reasonably short period of time. However, anatomic and immunologic differences between mice and humans mean that murine models have inherent limitations that must be considered when interpreting the results obtained from experimental models and applying these to the pathogenesis of CF disease in humans. This review will focus on the different CF mouse models available that represent diverse phenotypes observed in humans with CF and that can help researchers elucidate the diverse functions of the CFTR protein.     

Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g., NOD/SCID/γc^null and Rag2^nullγc^null mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.