Antigen mAb Research Articles

Brief Intraperitoneal Radioimmunotherapy of Small Peritoneal Carcinomatosis Using High Activities of Noninternalizing 125I-Labeled Monoclonal Antibodies

We assessed the efficiency and toxicity of brief intraperitoneal radioimmunotherapy using high activities of (125)I-labeled monoclonal antibody (mAb) in the treatment of small-volume peritoneal carcinomatosis. Brief intraperitoneal radioimmunotherapy consisted of a 185-MBq (740 MBq/mg) intraperitoneal injection of (125)I-35A7 (an anti-carcinoembryonic antigen mAb) into athymic nude mice 4 d after peritoneal tumor xenografting and, after 1 h, abundant washing of the peritoneal cavity with saline solution to remove unbound radioactivity. Another group of mice received this treatment plus a 37-MBq intravenous injection of (125)I-35A7 on day 7 or 11 after grafting. Control groups received a brief treatment followed by an additional intravenous injection on day 7 of either saline solution or irrelevant (125)I-PX. Tumor growth was monitored by bioluminescence imaging and SPECT/CT, and hematologic toxicity was evaluated by complete blood counts. Survival time was reported, and the mice were sacrificed when the bioluminescence signal reached 4.5 × 10(7) photons/s. The biodistribution of (125)I-35A7 mAb after intravenous or brief treatment was assessed, and the mean absorbed irradiation dose by organs and tumors was calculated using the MIRD formalism. Mild, transient hematologic toxicity was observed after the brief treatment plus intravenous (125)I-mAb, with no weight loss. Median survival increased from 32 d in the control groups, to 46 d in the brief treatment group, to 66 d in the group additionally receiving intravenous treatment on day 11, to 73 d in the group additionally receiving intravenous treatment on day 7. The brief treatment alone resulted in a 3-fold higher tumor-to-blood uptake ratio than did the standard intravenous treatment, and the mean absorbed irradiation doses by tumors were 11.6 Gy for the brief treatment and 16.7 Gy for the additional intravenous treatment. For healthy tissues other than blood, the mean absorbed irradiation dose did not exceed 1 Gy after brief treatment and 4.2 Gy after intravenous treatment. The efficiency, low toxicity, and high tumor-to-healthy tissue uptake ratio associated with brief intraperitoneal (125)I-35A7 radioimmunotherapy suggest that this method can be used in combination with radiation-synergistic drugs in the therapy of small-volume peritoneal carcinomatosis after cytoreductive surgery.

HIGH DOSES OF TCR STIMULI PREVENT CONVERSION OF NAÏVE CD4+ T CELLS INTO iTREGS IN AN NF-kB-DEPENDENT MANNER (139.10)

Abstract Conversion of naïve CD4+ T cells into FoxP3-expressing regulatory T cells (iTregs) can be achieved in the presence of TGF-b and IL-2 upon TCR triggering by intermediate concentrations of antigen or anti-CD3 mAb, but is prevented by high concentration of antigen. The mechanism for this high dose inhibition of FoxP3 induction has not been elucidated. As TCR stimulation promotes NF-kB signaling, we hypothesized that TCR-induced NF-kB may be responsible for inhibition of FoxP3 expression at high TCR occupancy. Whereas increasing doses of anti-CD3 mAb or antigenic peptide resulted in inhibition of FoxP3 induction in wildtype T cells under iTreg conditions, CD4 transgenic cells expressing an NF-kB superrepressor retained the ability to express FoxP3. Conversely, overactivation of the NF-kB pathway via a constitutively active IKKb reduced Foxp3 expression in wildtype T cells at lower TCR occupancy. Furthermore, CARMA1, a key molecule for NF-kB signaling downstream of the TCR, was critical for Foxp3 inhibition at high TCR occupancy. This was not due to increased TGF-b- or IL-2-mediated signals, or diminished Akt activation in CARMA1-KO T cells, as phosphorylation of Smad2, STAT5 and the Akt target S6 was similar to that in wildtype cells. Rather, the accessibility of the Foxp3 locus was impaired in wildtype but not CARMA1-deficient T cells. Our results suggest that higher levels of NF-kB can inhibit FoxP3 chromatin remodeling, thereby preventing its induction.

Generation and Characterization of Four Novel Monoclonal Antibodies Against Human Programmed Death-1 Molecule

Programmed death-1 (PD-1, CD279), an inhibitory co-stimulatory molecule of the CD28 superfamily, plays a critical role in immune response. In this report, four novel mouse anti-human PD-1 monoclonal antibodies (MAbs) were prepared using hybridoma technology and immunological characteristics of the MAbs were determined. The results showed that all the MAbs (clones 9H1, 4B9, 8F5, and 1F8) were IgG1(kappa) and bound specifically to human PD-1. By mutual competition, we found that the antibodies recognized three different epitopes of PD-1 antigen and 9H1 MAb could block both PD-1/PD-L1 and PD-1/PD-L2 interaction. Cross-linking of PD-1 with MAb 9H1 markedly blocked PD-1 negative signal and promoted T cell proliferation. In addition, 4B9 and 9H1 MAbs were suitable for indirect ELISA detection. Thus, the MAbs against human PD-1 with high specificity and different activity would be useful for the further study of this molecule.

Immunoassays for the Rapid Detection of Gentamicin and Micronomicin in Swine Muscle

A monoclonal antibody (mAb)-based ELISA and strip test for gentamicin (GEN) and its analogue micronomicin (MIN), are reported in this study. The conjugate gentamicin-glutaraldehyde-bovine serum albumin (GEN-GDA-BSA) was used as an immunogen. The produced anti-GEN mAB exhibited high cross-reactivity with micronomicin (MIN; 131.2%) and slight or negligible crossreactivity with other aminoglycosides. Based on this mAB, an ELISA and a strip test for GEN and MIN were developed and evaluated. The ELISA showed a 50% inhibition concentration (IC50) of 0.75 ng/mL for GEN and 0.58 ng/mL for MIN. For GEN, the average recoveries at 25-200 microg/kg ranged from 73 to 91%, with intraday CVs of 9-16% and interday CVs of 8-15%. For MIN, the average recoveries ranged from 108 to 131%, with intraday CVs of 10-16% and interday CVs of 8-15%. In contrast, the strip test for GEN or MIN had a detection limit of 5 ng/mL in phosphate-buffered saline and 50 microg/kg in muscle (n=24), and the results could be judged within 10 min. The detection results of incurred samples analyzed by the strip test, ELISA, and HPLC indicated that the two immunoassays correlated well with the HPLC method and could be used as convenient tools for the rapid screening of GEN and MIN residues in swine muscle.

Development of monoclonal antibodies for analyzing immune and hematopoietic systems of common marmoset

Common marmosets are considered experimental animals of primates useful for medical research. We developed several monoclonal antibodies (mAbs) directed to CD molecules to gain initial insight into the immune and hematopoietic systems of this organism, and analyzed the basic cellularity and characters of marmoset lymphocytes. Anti-marmoset CD antigen mAbs were prepared using marmoset antigen-expressing transfectants and used for flow cytometric analyses and cell fractionation. Expression of T-cell-related cytokine gene transcripts was examined in response to T-cell receptor stimulation by reverse transcription polymerase chain reaction analyses. Hematopoietic progenitor activities of marmoset bone marrow cells were examined in fractionated cells by mAbs against CD117 (c-kit) and CD34. CD4 and CD8 expression profiles in T-cell subsets of marmoset were essentially similar to those in mouse and human. CD4(+) and CD8(+) subsets were isolated from marmoset spleens. Detected transcripts after stimulation of T cells included Th1-, Th2-, and Th17-related cytokines in CD4(+) cells and cytotoxic proteases in CD8(+) cells, respectively. Colony-forming abilities were detected mainly in CD117 (c-kit)(+) cells, irrespective of CD34 expression. Marmoset immune system was basically similar to human and mouse systems.

Radiolabeled Antibodies to Bacillus anthracis Toxins Are Bactericidal and Partially Therapeutic in Experimental Murine Anthrax

Bacillus anthracis is a powerful agent for use in biological warfare, and infection with the organism is associated with a high rate of mortality, underscoring the need for additional effective therapies for anthrax. Radioimmunotherapy (RIT) takes advantage of the specificity and affinity of the antigen-antibody interaction to deliver a microbicidal radioactive nuclide to a site of infection. RIT has proven therapeutic in experimental models of viral, bacterial, and fungal infections; but it is not known whether this approach can successfully employ toxin binding monoclonal antibodies (MAbs) for diseases caused by toxigenic bacteria. Indirect immunofluorescence studies with MAbs to protective antigen (MAbs 7.5G gamma2b and 10F4 gamma1) and lethal factor (MAb 14FA gamma2b) revealed the surface expression of toxins on bacterial cells. Scatchard analysis of MAbs revealed high binding constants and numerous binding sites on the bacterial surface. To investigate the microbicidal properties of these MAbs, our group radiolabeled MAbs with either (188)Re or (213)Bi. In vitro, (213)Bi was more efficient than (188)Re in mediating microbicidal activity against B. anthracis. The administration of MAbs [(213)Bi]10F4 gamma1 and [(213)Bi]14FA gamma2b prolonged the survival of A/JCr mice infected with B. anthracis Sterne bacterial cells but not B. anthracis Sterne spores. These results indicate that RIT with MAbs that target B. anthracis toxin components can be used to treat experimental anthrax infection and suggest that toxigenic bacteria may be targeted with radiolabeled MAbs.

Investigation of Blood Typing Method for Seoul National University Miniature Pig

Pig blood group antigens may be present on grafted tissue as 16 isoforms, including the major one, A substrance. Seoul National University (SNU) miniature pigs and domestic pigs were used in this study. Polymerase chain reaction (PCR) was performed for the erythrocyte antigen A (EAA) gene, and reverse transcriptase-PCR for pig A transferase fucosyl transferase (FUT) 1 and FUT-2. The hemagglutination test was performed with murine monoclonal anti-A and anti-B antibodies (mAb), and immunohistochemistry with anti-human blood antigen mAb. SNU miniature and domestic pigs showed blood groups A and O. Blood group A SNU miniature pigs expressed either EAA AA or EAA AO and either S SS or S SO; blood group O miniature pigs expressed EAA OO and S SS or S SO, and there was no A weak. Additionally, blood group A could be divided into blood group A clotting and blood group A not clotting in hemagglutination tests. Pig A substance was expressed in the lung and kidney in blood group A pigs, but we could not detect pig A substance expression in the lung, kidney, and heart of blood group O pigs or the heart of blood group A pigs. In conclusion, we suggest that blood typing of SNU miniature pigs can be easily performed using immunohistochemistry, PCR, and/or RT-PCR. Molecular-based AO typing described in this study may be useful to select SNU miniature pigs bearing a specific blood group.

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice

We have studied cellular components required for xenograft survival mediated by anti-CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4(+) but not CD8(+) cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade-induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jalpha18(null), CD1(null), or beta2 microglobulin (beta2M)(null) NK 1.1 depleted, or perforin(null). Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti-CD154 mAb treatments. Additional groups received mAb or cells only. We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jalpha18 component of the T-cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jalpha18(null) and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jalpha18(null) recipients. These data imply a role for Jalpha18(+) NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1(null) mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1(+) cells adversely affects rat skin graft survival. An additional assay in beta2M(null) mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin-secreting NK cells. We conclude that MHC class I(+) CD1(+) Jalpha18(+) NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti-CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.

Anti-HIV-1 activities of 4 Telomerase Restrictors

MTT Cell Proliferation Assay was used to optimize the concentration of Telomerase Restrictors(TRs) with minimum toxicity to the selected cells. FACSort flow cytometer and Innotest P24 HIV(Human immunodeficiency Virus) antigen mAb ELISA Kit were used to investigate the anti-HIV-1 activities of TRs. The results showed that TRs had low cytotoxicity to the PBMC (Peripheral Blood mononuclear cells) and CEM/GFP if the concentration of TRs was at 50 μmol/L or below, and the supernatant from PBMC pretreated with SHIV and TR1-001 /TR1-002 could not infect the PBMC, while can infect the C8166 with reduced infectivity, which suggested that the TRs may be one of the novel resources for screening anti-HIV-1 agents.

Tumor immunolocalization using 124I-iodine-labeled JAA-F11 antibody to Thomsen–Friedenreich alpha-linked antigen

Clinical immunolocalization has been attempted by others with an anti-Thomsen–Friedenreich antigen (TF-Ag) mAb that bound both alpha- and beta-linked TF-Ag. In this report, 124I-labeled mAb JAA-F11 specific for alpha-linked TF-Ag showed higher tumor specificity in in vivo micro-positron emission tomography (micro-PET) of the mouse mammary adenocarcinoma line, 4T1, showing no preferential uptake by the kidney. Labeled product remained localized in the tumor for at least 20 days. Glycan array analysis showed structural specificity of the antibody.

Gangliosides, Ab1 and Ab2 antibodies: II. Light versus heavy chain: An idiotype-anti-idiotype case study

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti- N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

A Rapid and EfficientIn VivoMethod for Determining the Biologic Efficacy of Monoclonal Antibodies in Animal Models of Cancer

The selection of efficacious anti-tumor monoclonal antibodies (MAbs) for biological applications is a lengthy and labor-intensive process. In vitro characterization of one hybridoma fusion may reveal large numbers of tumor antigen-specific hybridomas. Very often, many of these tumor-specific antibodies need to be assessed in vivo using several different murine xenograft tumor prevention models to determine biological efficacy. The production and purification of sufficient quantities of many antigen-specific hybridomas is time-consuming, and several months can pass between initial determination of MAb specificity and bioactivity. Moreover, many tumor-specific MAbs selected using in vitro binding studies have no in vivo anti-tumor efficacy. These studies describe an in vivo screening method either to eliminate non-efficacious MAbs or to rank-order several tumor-specific MAbs in an expeditious manner. Proof-of-concept studies were conducted using two hybridomas secreting fully characterized neutralizing human anti-tumor MAbs (CNTO MAbs). Nu-/nu- mice were injected with CNTO MAb-secreting hybridoma cells in Matrigel cell matrix, followed by injection of target human tumor cells 4 days later (when circulating CNTO MAbs were detected in serum). Both the tumor take-rate and the mean tumor volumes were reduced significantly in mice treated with CNTO MAbsecreting hybridomas compared with mice treated with non-antibody-secreting cells. A panel of human antitumor antigen-specific MAbs with unknown biological efficacy was then evaluated by this method. The hybridomas exhibited a varied pattern of anti-tumor protection, indicating that some hybrids were secreting neutralizing anti-tumor MAbs, while others appeared to be less efficacious. These studies demonstrate a rapid, biologically relevant "yes/no" in vivo screening method for the evaluation of anti-tumor antigen MAbs.

HLA Class I Antigen Down-Regulation in Primary Ovary Carcinoma Lesions: Association with Disease Stage

To investigate TAP1, TAP2, and HLA class I antigen expression in primary ovarian carcinoma lesions and to assess the clinical significance of defects in the expression of these molecules. Fifty-one formalin-fixed, paraffin-embedded primary ovarian carcinoma lesions were stained with affinity-purified rabbit anti-TAP1 and anti-TAP2 antibodies and with anti-HLA class I heavy chain monoclonal antibody (mAb) HC-10 using the immunoperoxidase reaction. The results of immunohistochemical staining were correlated with the histopathologic characteristics of the lesions and with patients' survival. Ovarian surface epithelium, thecal cells of follicles, and stromal cells were stained by anti-TAP1, anti-TAP2, and anti-HLA class I antigen xenoantibodies with a homogeneous pattern. In contrast, no staining of lutheinic cells by these antibodies was detected. Forty-one and 32 out of 51 primary ovarian carcinoma lesions were stained by anti-TAP1 and anti-TAP2 xenoantibodies and by anti-HLA class I antigen mAb HC-10, respectively. The staining patterns by anti-TAP1 and anti-TAP2 xenoantibodies were completely concordant, but did not correlate with that by anti-HLA class I heavy chain mAb HC-10. TAP1 and TAP2 expression was associated neither with the histopathologic characteristics of the lesions nor with clinical variables. On the other hand, HLA class I antigen down-regulation was associated with disease stage: the odds ratio of stage III for HLA class I antigen negative patients was 7.6 (95% confidence interval, 1.9-30.5; P= 0.007), whereas for TAP negative patients was 5.1 (95% confidence interval, 0.9-28.4; P = 0.07). Follow up was available for 39 out of the 51 patients. Multivariate analysis showed that both grading and staging were associated with a higher risk of death, whereas TAP and HLA class I antigen phenotypes were not. The lack of association between TAP and HLA class I antigen expression is compatible with the possibility that multiple mechanisms underlie HLA class I antigen down-regulation in primary ovarian carcinoma lesions. The potential role of immunologic events in the clinical course of ovarian carcinoma suggests that the association between HLA class I antigen down-regulation and disease progression may reflect the escape of tumor cells from immune recognition and destruction.

SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity

The humanization of mAbs by complementarity-determining region (CDR)-grafting has become a standard procedure to improve the clinical utility of xenogeneic Abs by reducing human anti-murine Ab (HAMA) responses elicited in patients. However, CDR-grafted humanized Abs may still evoke anti-V region responses when administered in patients. To minimize anti-V region responses, the Ab may be humanized by grafting onto the human templates only the specificity-determining residues (SDRs), the residues that are essential for the surface complementarity of the Ab and its ligand. Typically, humanization of an Ab, whether by CDR or SDR grafting, involves the use of a single human template for the entire VL or VH domain of an Ab. We hypothesized, however, that the homology between the human template sequences and mAb to be humanized may be maximized by using templates from multiple human germline sequences corresponding to the different segments of the variable domain. This could be more advantageous in reducing the potential immunogenicity of the humanized Ab. This report describes the SDR grafting of the murine anti-carcinoembryonic antigen (CEA) mAb COL-1 using three different human germline V-kappa sequences as templates for the VL CDRs and another human template for the VL frameworks. In competition RIAs, the SDR-grafted COL-1 (HuCOL-1 SDR) completely inhibited the binding of radiolabeled murine COL-1 (mCOL-1) to CEA, and showed that its binding affinity is comparable to that of the CDR-grafted Ab (HuCOL-1). The HuCOL-1 SDR showed similar binding reactivity to the CEA expressed on the surface of a tumor cell line as the HuCOL-1. More importantly, compared to HuCOL-1 and the “abbreviated” CDR-grafted Ab, HuCOL-1 SDR showed lower reactivity to patients’ sera carrying anti-V region Abs to mCOL-1. HuCOL-1 SDR, which shows a lower sera reactivity than that of the parental Abs while retaining its Ag-binding property, is a potentially useful clinical reagent. To the best of our knowledge, this is the first time a VL or VH domain of an Ab has been humanized by grafting the SDRs onto a human template comprised of several Ab sequences. We have shown that humanization of an Ab can be optimized using multiple human templates for a single variable domain of an Ab. This approach maximizes the homology between the target Ab and the human templates in both the frameworks and the CDRs by choosing as the template the human sequence that displays the highest local sequence identity to the frameworks and to each of the CDRs of the target Ab.

Electrophoretic analysis of the cleaved form of serpin, squamous cell carcinoma antigen-1 in normal and malignant squamous epithelial tissues.

The aim of this study was to detect the cleaved form of serine proteinase inhibitor (serpin), squamous cell carcinoma (SCC) antigen-1 in normal and malignant squamous epithelial tissues, which implies the presence of its target proteinase. The cleaved SCC antigen-1 in normal squamous epithelium was identified as a single spot with pI 6.35 and M(r) 40,000 by two-dimensional electrophoresis (2-DE) combined with immunoblotting. Interestingly, the cleaved form showed different biochemical properties in heat stability or immunoreactivity with a monoclonal antibody for SCC antigen (Mab 426) compared to intact SCC antigen-1. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of tissue extracts showed an abundant 40 kDa band of cleaved SCC antigen-1 in tumor tissue compared to normal tissue. Among the potential target proteinase of SCC antigen-1, immunoblotting analyses revealed that cathepsin L2 was remarkably overexpressed in tumor tissue, while cathepsin L was expressed in both normal and tumor tissues. These findings indicate that SCC antigen-1 interacts with specific endogenous proteinases such as cathepsins L and L2 in physiological and pathological states of squamous epithelium.

Study of the Mechanism Involved in Angiogenesis and Synovial Cell Proliferation in Human Synovial Tissues of Patients with Rheumatoid Arthritis Using SCID Mice

To examine whether synovial cell proliferation is due to angiogenesis, we studied the relationship between the inhibition of synovial cell proliferation and an angiogenesis inhibitor, TNP-470, in human synovial tissues. Human synovial tissues were implanted into the back of SCID mice (SCID-HuAg mice). Sixteen mice were divided into two groups of eight mice each: the untreated group (vehicle group) and the TNP-470-treated group that received a dose of 10 mg/kg body weight by subcutaneous injection. The number of blood vessels and synovial lining cells clearly increased in the vehicle group, but the number of synovial lining cells clearly decreased and the blood vessels were hardly detected in the TNP-470 group. Immunohistochemically, cells that stained positively for the anti–proliferating cell nuclear antigen (PCNA) mAb were abundant in synovial lining cells and endothelial cells in synovial tissues. Cells that stained positively for the anti-CD34 polyclonal antibody were abundant in the endothelial cells in the vehicle group, but these positively stained cells were hardly detected in the TNP-470 group. The PCNA positivity ratio in the vehicle group was 0.64 ± 0.019, whereas that in the TNP-470 group was 0.199 ± 0.007. The numbers of cells that stained positively for anti-CD34 polyclonal antibody were 242 ± 13.4 in the vehicle group and 153 ± 6.73 in the TNP-470 group per 10 microscopic fields. Cells that stained positively for anti-mouse CD31 mAb were mainly localized in the synovial lining, but invaded the subsynovial lining layer in human synovial tissues. On the other hand, cells that stained positively for anti-human CD31 mAb were mainly localized in the subsynovial lining layer. We found that endothelial cell proliferation is dependent on angiogenesis based on the result that angiogenesis and synovial cell proliferation were inhibited by treatment with TNP-470.

Pathways of anaphylaxis in the mouse

Background: Although anaphylaxis is classically mediated by IgE, FcϵRI, mast cells, and histamine, several rodent studies suggest that an alternative pathway involving IgG, FcγRIII, macrophages and platelets, and platelet-activating factor (PAF) may be more important in the anaphylactic response to antigen challenge. Objectives: We sought to determine the relative roles of the classical and alternative pathways of anaphylaxis in a mouse model characterized by mastocytosis and a high level of antigen-specific IgE antibody. Methods: Wild-type, IgE-deficient, FcϵRI-deficient, and mast cell–deficient mice were immunized with goat anti-mouse IgD antibody, which induces mastocytosis and a large IgE and IgG anti-goat IgG response, and then challenged 14 days later with antigen (goat IgG) or rat anti-mouse IgE mAb. Specific vasoactive mediators, cell types, Ig isotypes, or Ig receptors were blocked or eliminated before challenge in some experiments. The severity of anaphylaxis was gauged by changes in body temperature, physical activity, and mortality. Results: Equal doses of antigen or anti-IgE mAb induced similar anaphylactic responses. Anti-IgE mAb-induced anaphylaxis was FcϵRI and mast cell dependent and mediated predominantly by histamine. In contrast, neither mast cells nor platelets appeared important for antigen-induced anaphylaxis, which was FcγRIII and macrophage dependent and mediated predominantly by PAF. Conclusions: Antigen-induced anaphylaxis in the mouse proceeds primarily through the IgG, FcγRIII, macrophage, and PAF pathway, even in an experimental model that is characterized by strong mast cell and IgE responses. The presence of FcγRIII on human macrophages makes it possible that the IgG, FcγRIII, macrophage, and PAF pathway also contributes to human anaphylaxis. (J Allergy Clin Immunol 2002;109:658-68.)

Evaluation of four HIV antigen tests

A direct comparison of different HIV antigen assays is very helpful in making an informed choice, not only for the testing laboratories but also for healthcare workers in the developing world who are looking for reliable and inexpensive tests/methods in the follow-up of their treated patients. As a follow-up to the study published previously [Fransen K., Martens G., Stynen D., Goris A., Nys P., Nkengasong J., Heyndrickx L., Janssens W., van der Groen G., 1997. J. Med. Virol. 53, 31–35] where only two tests have been compared, four different commercial methods for HIV antigen determination in plasma and supernatant of cell cultures have now been evaluated on a limited sample size (88): COULTER™ HIV-1 p24 Antigen Assay (Coulter), (Test 1) INNOTEST HIV Antigen mAb (Innogenetics) (Test 2), Genetic Systems™ HIV-1 Ag EIA (Sanofi-Pasteur 1 The company Sanofi-Pasteur is now referred to as Biorad. 1 ) (Test 3) and VIDAS HIV P24 II (bioMérieux) (Test 4). Of the four tests used in this study, Test 2 was by far the most sensitive test. In a population of 88 follow-up samples from 35 different patients representing all stages of infection, the test detected confirmed p24 antigen at least once in 85.7% (30/35) of these patients, versus Test 3 in 74.3% (26/35), Test 4 in 71.4% (25/35), and Test 1 in 48.6% (17/35) of the patients. Test 2 detected confirmed p24 antigen in 84.9% of the follow-up samples, followed by Test 4 (65.9%), Test 3 (64.8%) and Test 1 (39.8%). Finally, Test 2 also proved best for detecting genetically diverse isolates.

Immunohistochemical and Reverse Transcription-Polymerase Chain Reaction Expression Analysis of Tyrosinase and Microphthalmia-Associated Transcription Factor in Angiomyolipomas

Angiomyolipomas (AMLs) show a characteristic immunoreactivity with melanocyte differentiation markers such as monoclonal antibody (mAb) HMB45, which detects melanocyte differentiation antigen gp100 and mAb A103 reacting with Melan-A/MART-1. Monoclonal antibody T311 to tyrosinase (a key enzyme of melanogenesis) and mAb D5 to the microphthalmia (Mitf) antigen are two newly available markers of melanocytic differentiation. The authors tested 15 AMLs with T311 and D5 by immunohistochemistry and a subset of 3 cases by reverse transcription-polymerase chain reaction for their expression of tyrosinase and Mitf mRNA. T311 showed poor sensitivity in AMLs because only focal staining was seen in 1 out of 15 cases, although tyrosinase mRNA was found in all tested cases. Mitf mRNA was present in 3 of 3 tested cases, and D5 was positive in 15 of 15 AMLs. However, D5 immunostaining often was focal and not as homogeneous as A103, which was analyzed in a previous study. D5 staining also could be seen in other cell types such as normal renal tubular cells, macrophages, and renal cell carcinoma. The current results show that in contrast with HMB45 and A103, T311 has little or no value in the diagnosis of AMLs. D5 may be useful in a panel of antibodies in the diagnosis of AMLs.

Time-resolved fluorescence imaging for specific and quantitative immunodetection of human kallikrein 2 and prostate-specific antigen in prostatic tissue sections

Objectives. To design protocols for specific and quantitative immunohistochemical detection of human kallikrein 2 (hK2) using lanthanide chelate-labeled monoclonal antibodies (Mabs) and time-resolved fluorescence imaging. Methods. Anti–prostate-specific antigen (PSA) Mabs were tested in microtiterplate assays for their ability to prevent PSA from cross-reacting with the anti-hK2 Mab 6H10. Europium-labeled 6H10 and terbium-labeled anti-PSA Mab 2E9, selected as the best blocker antibody, were used for dual-label immunodetection in routinely fixed benign (n = 7) and malignant (n = 5) prostate specimens. The amounts of IgG bound in tissue were calculated from drops containing known Mab concentrations. Results. The use of anti-PSA Mab 2E9 for blocking diminished the cross-reaction from 5% to 0.3%. In the analyzed tissues, there was considerable variation in staining intensity for both proteins; PSA signals varied from 0.1 to 36.6 times that of hK2, with on average 10-fold more bound anti-PSA Mab than anti-hK2 Mab. In malignant tissue, the amounts of bound IgGs were lower and more variable than in benign tissue using both the anti-PSA Mab and the anti-hK2 Mab. The variation in signal intensities for PSA and hK2 correlated significantly in benign tissue ( P >0.05), but not in benign hyperplastic and malignant specimens ( P <0.05). Conclusions. Quantification of two lanthanide chelate-labeled antibodies bound in the same tissue section enabled comparison of PSA and hK2 content in individual cells. The average cellular content of hK2 relative to that of PSA was consistent with previous mRNA studies. The time-resolved fluorescence imaging-based quantification method has universal applicability in fixed tissue specimens.