Variability of Complement Response toward Preclinical and Clinical Nanocarriers in the General Population.

Opsonization (coating) of nanoparticles with complement C3 component is an important mechanism that triggers immune clearance and downstream anaphylactic and proinflammatory responses. The variability of complement C3 binding to nanoparticles in the general population has not been studied. We examined complement C3 binding to dextran superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoworms, SPIO NWs, 58 and 110 nm) and clinically approved nanoparticles (carboxymethyl dextran iron oxide ferumoxytol (Feraheme, 28 nm), highly PEGylated liposomal doxorubicin (LipoDox, 88 nm), and minimally PEGylated liposomal irinotecan (Onivyde, 120 nm)) in sera from healthy human individuals. SPIO NWs had the highest variation in C3 binding (n = 47) between subjects, with a 15-30 fold range in levels of C3. LipoDox (n = 12) and Feraheme (n = 18) had the lowest levels of variation between subjects (an approximately 1.5-fold range), whereas Onivyde (n = 18) had intermediate between-subject variation (2-fold range). There was no statistical difference between males and females and no correlation with age. There was a significant correlation in complement response between small and large SPIO NWs, which are similar structurally and chemically, but the correlations between SPIO NWs and other types of nanoparticles, and between LipoDox and Onivyde, were not significant. The calculated average number of C3 molecules bound per nanoparticle correlated with the hydrodynamic diameter but was decreased in LipoDox, likely due to the PEG coating. The conclusions of this study are (1) all nanoparticles show variability of C3 opsonization in the general population; (2) an individual's response toward one nanoparticle cannot be reliably predicted based on another nanoparticle; and (3) the average number of C3 molecules per nanoparticle depends on size and surface coating. These results provide new strategies to improve nanomedicine safety.

IL-12hi rapamycin-conditioned dendritic cells mediate IFN-γ-dependent apoptosis of alloreactive CD4+ T cells in vitro and reduce lethal graft-versus-host disease.

Rapamycin (RAPA) inhibits the mechanistic target of rapamycin (mTOR), a crucial immune system regulator. Dendritic cells (DC) generated in RAPA (RAPA-DC) enrich for CD4(+) forkhead box p3 (FoxP3(+)) regulatory T cells and induce T cell apoptosis by an unknown mechanism. RAPA-DC also promote experimental allograft survival, yet paradoxically secrete increased IL-12, crucial for the generation of IFN-γ(+) CD4(+) T cells. However, IFN-γ is pro-apoptotic and IL-12-driven IFN-γ inhibits experimental graft-versus-host disease (GVHD). We hypothesized that IL-12(hi) RAPA-DC would facilitate IFN-γ-mediated apoptosis of alloreactive T cells and, unlike control (CTR)-DC, would reduce lethal GVHD. Following LPS stimulation, RAPA-DC exhibited decreased MHCII and co-stimulatory molecules and contained a significant population of CD86(lo) IL-12(hi) cells. Consistent with our hypothesis, both unstimulated and LPS-stimulated RAPA-DC enhanced alloreactive CD4(+) T cell apoptosis in culture. Augmented T cell apoptosis was ablated by IFN-γ neutralization or using T cells lacking the IFN-γ receptor, and it was associated with increased expression of Fas and cleaved caspase 8. DC production or responses to IFN-γ were not important to increased apoptotic functions of RAPA-DC. LPS-stimulated IL-12p40(-/-) RAPA-DC induced lower levels of T cell apoptosis in culture, which was further decreased with addition of anti-IFN-γ. Finally, whereas CTR-DC accelerated mortality from GVHD, LPS-treated RAPA-DC significantly prolonged host survival. In conclusion, increased apoptosis of allogeneic CD4(+) T cells induced by LPS-stimulated IL-12(hi) RAPA-DC is mediated in vitro through IFN-γ and in part by increased IL-12 expression. Enhanced production of IL-12, the predominant inducer of IFN-γ by immune cells, is a probable mechanism underlying the capacity of LPS-treated RAPA-DC to reduce GVHD.

The immunomodulatory effects of pegylated liposomal doxorubicin are amplified in BRCA1--deficient ovarian tumors and can be exploited to improve treatment response in a mouse model.

OBJECTIVE: Women with BRCA-associated ovarian cancer demonstrate excellent responses to Pegylated Liposomal Doxorubicin (PLD). PLD has also been shown to enhance T cell recognition of tumor cells. Here we characterize immunophenotypic changes associated with BRCA1 dysfunction in ovarian cancer cells, and evaluate the T cell contribution to the therapeutic efficacy of PLD in a BRCA1- ovarian cancer model to determine whether enhanced anti-tumor immunity contributes to the improved response to PLD in BRCA1- ovarian cancers. METHODS: The immunophenotype of BRCA1- and wild-type (WT) ovarian cancer cells and their response to PLD were compared in vitro using flow cytometry. T cell recruitment to BRCA1- tumors was evaluated with flow cytometry and immunohistochemistry. The contribution of T cell populations to the therapeutic effect of PLD in a BRCA1- model was evaluated using immunodepleting antibodies with PLD in vivo. RESULTS: The cytotoxic response to PLD was similar in BRCA1- and WT cells in vitro. BRCA1- inactivation resulted in higher expression of Fas and MHC-I at baseline and after PLD exposure. PLD prolonged the survival of BRCA1- tumor bearing mice and increased intratumoral T cell recruitment. CD4+ depletion combined with PLD significantly prolonged overall survival (p=0.0204) in BRCA1- tumor-bearing mice. CONCLUSION: Differences in the immunophenotype of BRCA1- and WT cells are amplified by PLD exposure. The enhanced immunomodulatory effects of PLD in BRCA1- tumors may be exploited therapeutically by eliminating suppressive CD4+ T cells. Our results support further study of combination therapy using PLD and immune agents, particularly in women with BRCA gene mutations.

Increased immunogenicity of surviving tumor cells enables cooperation between liposomal doxorubicin and IL-18.

BACKGROUND: Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated. METHODS: Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack in vitro. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated in vivo in mice bearing ID8-Vegf tumors. RESULTS: While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death in vitro. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy in vivo and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice. CONCLUSION: These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 in vivo.

Reprogramming the T cell response to cancer by simultaneous, nanoparticle-mediated PD-L1 inhibition and immunogenic cell death.

In this study, dual drug-loaded nanoparticles were constructed to co-deliver low-dose doxorubicin (DOX) and miR-200c (DOX/miR-NPs) to inhibit programmed death-1 receptor (PD-L1) expression and trigger immunogenic cell death (ICD) in cancer cells. Two block copolymers, folic acid (FA)-conjugated PLGA-PEG (PLGA-PEG-FA) and PLGA-PEI, were formulated as folate-targeted NPs and loaded with DOX and miR-200c. The NPs, which were formed as nanosize objects (110.4 ± 2.1) with narrow size distribution (0.19 ± 0.02), effectively protected the miR-200c from degradation in serum. Modifying the NPs with FA increased not only their uptake by cancer cells in vitro but also their accumulation in tumor microenvironments in vivo, as compared with those properties of non-FA-modified NPs. The DOX/miR-NPs also exhibited efficacious inhibition of PD-L1 expression and robust induction of ICD in cancer cells in vitro and in vivo, resulting in increased dendritic cell maturation and CD8+ T cell response towards cancer cells. Furthermore, tumor growth was significantly inhibited by folate-targeted NPs loaded with the low-dose DOX/miR-200c combination, but not by treatments with free DOX, miR-NPs or DOX-NPs. Thus, our results suggest that simultaneous PD-L1 inhibition via microRNAs and the induction of an immunogenic tumor microenvironment via low-dose cytotoxic drugs may improve cancer therapy efficacy.

Pre-existing anti-polyethylene glycol antibody reduces the therapeutic efficacy and pharmacokinetics of PEGylated liposomes.

Rationale: Increasing frequency of human exposure to PEG-related products means that healthy people are likely to have pre-existing anti-PEG antibodies (pre-αPEG Ab). However, the influence of pre-αPEG Abs on the pharmacokinetics (PK) and therapeutic efficacy of LipoDox is unknown. Methods: We generated two pre-αPEG Ab mouse models. First, naïve mice were immunized with PEGylated protein to generate an endogenous αPEG Ab titer (endo αPEG). Second, monoclonal αPEG Abs were passively transferred (αPEG-PT) into naïve mice to establish a αPEG titer. The naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its PK. Tumor-bearing naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its biodistribution. The therapeutic efficacy of LipoDox was estimated in the tumor-bearing mice. Results: The areas under the curve (AUC)last of LipoDox in endo αPEG and αPEG-PT mice were 11.5- and 15.6- fold less, respectively, than that of the naïve group. The biodistribution results suggested that pre-αPEG Ab can significantly reduce tumor accumulation and accelerate blood clearance of 111In-labeled LipoDox from the spleen. The tumor volumes of the tumor-bearing endo αPEG and αPEG-PT mice after treatment with LipoDox were significantly increased as compared with that of the tumor-bearing naïve mice. Conclusions: Pre-αPEG Abs were found to dramatically alter the PK and reduce the tumor accumulation and therapeutic efficacy of LipoDox. Pre-αPEG may have potential as a marker to aid development of personalized therapy using LipoDox and achieve optimal therapeutic efficacy.

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate.

AIM: To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS: The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM). RESULTS: Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner. CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

Accelerated blood clearance of PEGylated liposomes upon repeated injections: effect of doxorubicin-encapsulation and high-dose first injection.

The "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection, has been reported to occur in rodents and rhesus monkeys. This rapid clearance was reported to be caused by the binding of PEG-specific IgM, which was generated by the first dose of injected liposomes, to the second dose of liposomes and the subsequent activation of complement, serving in turn as an opsonin. Although there are several PEGylated liposomal formulations, such as Doxil/Caelyx loaded with doxorubicin (DXR), in clinical use, the rapid clearance phenomenon has never been reported for such formulations. In the present article, we report that a first injection of PEGylated liposomes containing encapsulated DXR failed to induce the ABC phenomenon. Likewise, no rapid clearance of the test dose was observed when the first dose of "empty" PEGylated liposomes (without DXR) exceeded 5 micromol phospholipids/kg. By contrast, "empty" PEGylated liposomes at a low dose (1 micromol phospholipids/kg) induced the phenomenon as before. Western blot analysis revealed abundant binding of IgM to PEGylated liposomes when these were incubated in serum from rats that had received "empty" PEGylated liposomes. Substantially less binding of IgM was found when the liposomes were incubated in serum from rats treated with DXR-loaded PEGylated liposomes. For both the empty and the DXR-containing liposomes the amounts of IgM binding to the liposomes decreased with an increasing dose of injected liposomes. Serum obtained from rats following injection of empty PEGylated liposomes caused complement activation by addition of PEGylated liposomes in an inversely dose-dependent manner: the lower the dose, the higher the complement activation. By contrast, no complement activation was detected with serum from rats that had been treated with DXR-loaded PEGylated liposomes. These findings suggest that encapsulation of DXR as well as a relatively high lipid dose abrogate the immune response against PEGylated liposomes which is observed with the same liposomes but without DXR and at low doses. Our observations may thus have important implications for the development, evaluation and therapeutic use of liposomal cytotoxic drug formulations requiring multiple injection schemes.

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Radioimmune assay and characteristics of antibodies to macromomycin (NSC 170105).

A radioimmune assay for the antitumor agent, macromomycin, using purified, radioiodine-labeled macromomycin and antisera raised in rabbits against a carbodiimide-catalyzed macromycin-Limulus polyphemus hemocyanin complex has been developed. Radiolabeled macromomycin was prepared by direct iodination of the polypeptide antibiotic with the use of iodine monochloride or solid-state lactoperoxidase. Antibody-bound drug was isolated from free macromomycin with dextran-coated, activated charcoal. The standard curve of the sequential saturation assay was linear on a logit-log plot and indicated a lower limit of sensitivity of approximately 100 pg macromomycin. The radioimmune assay was suitable for measuring macromomycin in the presence of other antitumor drugs, and detection of macromomycin was quantitative when it was added to normal human serum or urine. Drug binding to melanoma and mammary carcinoma cell surfaces could be inhibited by preincubating macromomycin with affinity-purified antimacromomycin antibodies. However, once the drug was bound to cell surfaces, addition of antimacromomycin antibodies did not result in removal of the drug from cell surfaces or in reversal of macromomycin-induced inhibition of thymidine incorporation into cellular DNA. Antimacromovide useful tools for developing pharmacokinetic and toxicity studies of macromomycin, as well as for analyzing the mechanism(s) of action of the drug.

Oral administration of anti-doxorubicin monoclonal antibody prevents chemotherapy-induced gastrointestinal toxicity in mice.

Gastrointestinal mucositis is a common and painful condition that afflicts a proportion of cancer patients receiving chemotherapeutic drugs including anthracyclines, and it has become the dose-limiting toxicity for a number of chemotherapeutic regimens. The murine monoclonal antibody MAD11 recognizes the anthracycline doxorubicin, and systemic administration of this antibody in mice treated with doxorubicin was found previously to prevent the toxic effects of the drug. The purpose of this study was to determine whether gastrointestinal toxicity associated with doxorubicin can be reduced by oral administration of anti-doxorubicin MAD11 in mice. Our experiments show that orally administered MAD11 antibodies: (a) are essentially not absorbed in the blood circulation since less than 0.5% of protein-associated radioactivity was recovered from blood samples; (b) reduce the extent of doxorubicin-induced apoptosis in murine intestinal crypts, as determined by labeling strand breaks with modified nucleotides in an enzymatic reaction; and (c) reduce the body weight loss in mice treated with 12 mg/kg body weight of doxorubicin and decrease the early mortality in mice treated with 16 mg/kg body weight. This type of treatment may be useful in preventing anthracycline-induced gastrointestinal mucositis in cancer patients.

An integrated assessment of morphology, size, and complement activation of the PEGylated liposomal doxorubicin products Doxil®, Caelyx®, DOXOrubicin, and SinaDoxosome.

In order to improve patient's benefit and safety, comprehensive regulatory guidelines on specificities of Non-Biological Complex Drugs (NBCDs), such as doxorubicin-encapsulated liposomes, and their follow-on versions are needed. Here, we compare Doxil® and its European analog Caelyx® with the two follow-on products DOXOrubicin (approved by the US Food and Drug Administration) and SinaDoxosome (produced in Iran) by cryogenic transmission electron microscopy, dynamic light scattering and Nanoparticle Tracking Analysis, and assess their potential in activating the complement system in human sera. We found subtle physicochemical differences between the tested liposomal products and even between the tested batches of Doxil® and Caelyx®. Notably, these included differences in vesicular population aspect ratios and particle number. Among the tested products, only SinaDoxosome, in addition to the presence of unilamellar vesicles with entrapped doxorubicin crystals, contained empty circular disks. Differences were also found in complement responses, which may be related to some morphological differences. This study has demonstrated an integrated biophysical and immunological toolbox for improved analysis and detection of physical differences among vesicular populations that may modulate their clinical performance. Combined, these approaches may help better product selection for infusion to the patients as well as for improved design and characterization of future vesicular NBCDs with enhanced clinical performance and safety.

Immunocytochemistry for drugs containing an aliphatic primary amino group in the molecule, anticancer antibiotic daunomycin as a model.

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH4, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues.

Rapamycin Impairs Antitumor CD8+ T-cell Responses and Vaccine-Induced Tumor Eradication.

The metabolic sensor mTOR broadly regulates cell growth and division in cancer cells, leading to a significant focus on studies of rapamycin and its analogues as candidate anticancer drugs. However, mTOR inhibitors have failed to produce useful clinical efficacy, potentially because mTOR is also critical in T cells implicated in immunosurveillance. Indeed, recent studies using rapamycin have demonstrated the important role of mTOR in differentiation and induction of the CD8+ memory in T-cell responses associated with antitumor properties. In this study, we demonstrate that rapamycin harms antitumor immune responses mediated by T cells in the setting of cancer vaccine therapy. Specifically, we analyzed how rapamycin affects the antitumor efficacy of a human papilloma virus E7 peptide vaccine (CyaA-E7) capable of eradicating tumors in the TC-1 mouse model of cervical cancer. In animals vaccinated with CyaA-E7, rapamycin administration completely abolished recruitment of CD8+ T cells into TC-1 tumors along with the ability of the vaccine to reduce infiltration of T regulatory cells and myeloid-derived suppressor cells. Moreover, rapamycin completely abolished vaccine-induced cytotoxic T-cell responses and therapeutic activity. Taken together, our results demonstrate the powerful effects of mTOR inhibition in abolishing T-cell-mediated antitumor immune responses essential for the therapeutic efficacy of cancer vaccines.

PSK as a chemopreventive agent.

PSK, a protein-bound polysaccharide preparation obtained from cultured mycelia of the CM-101 strain of Coriolus versicolor belonging to basidiomycetes, is a biological response modifier capable of exhibiting diverse biological activities. This agent has been used clinically for the treatment of postoperative cancer patients in Japan by oral use. In this paper, chemopreventive aspects of PSK were reviewed. Oral administration of PSK reduced the incidence of tumor and/or prolonged the survival period in the following chemical carcinogen-induced, radiation-induced, and spontaneously developed animal cancer models: rat gastrointestinal cancer induced by 1,2-dimethylhydrazine; rat hepatoma by 3'-methyl-dimethylaminobenzene; mouse thymic lymphoma by whole-body irradiation; mouse spontaneous mammary tumor; and so on. PSK did not interact and/or inhibit drug-metabolizing enzymes and had no effect on the Ames test. On the other hand, this agent scavenged active oxygen through the induction of manganese superoxide dismutase, prevented the increase in frequency of anticancer agent-induced sister chromatid exchange, and suppressed fetal deformation induced by transplacental injection of teratogen, suggesting an effect on the initiation or promotion process of carcinogenesis. Also, PSK regulated cytokine production and enhanced the antitumor activity of effector cells such as killer T-cells and natural killer cells, suggesting an effect on the growth process after the development of malignant cells. Thus, this agent seems to act at multiple steps during carcinogenesis rather than a particular step. The main mechanism may be an antiteratogenic effect attributed to radical trapping, preventive effects against chromosome injury, and immunomodulative effects attributed to the modulation of cytokine production and effector cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme immunoassay with high sensitivity and accuracy for specific antibody to neocarzinostatin.

A quantitative enzyme immunoassay (EIA) for specific antibody to neocarzinostatin (NCS) is described which uses enzyme-labeled anti-rabbit IgG antibody, solid-phase NCS and standard purified specific antibody to NCS. The dose of the standard was determined by sandwich EIA for rabbit IgG. The lower detection limit was 3 ng of the specific antibody per tube. The accuracy of the assay was excellent and a comparative study with the sandwich EIA for rabbit IgG showed good correlation. The antiserum to NCS of the highest titer was found to contain 0.6 mg and 40 mg per ml of specific antibody to NCS and of normal IgG, respectively. The accuracy of the assay results and the purity of the standard was established by 2 recovery tests for anti-NCS antibody.

Deoxyspergualin neither counteracts lipopolysaccharide (LPS) or Staphylococcus aureus enterotoxin-B (SEB) induced lethality in mice nor does it modulate the release of tumor necrosis factor-alpha.

To gain further insights into the immunopharmacological mode of action of the immunosuppressant antibiotic deoxyspergualin (DSP), its effects were evaluated in murine lethal endo- and exotoxemia. These are two cytokine-mediated macrophage and T cell dependent immunoinflammatory conditions that can be induced in D-Galactosamine (D-Gal) presensitized mice by the injections with either LPS or SEB, respectively. The results show that prophylactic treatment with DSP (2.5 or 5 mg/kg bd.wt. 48, 24 and 2 h prior to challenge) neither improved the rate of survival, nor influenced the massive increase in the blood levels of tumor necrosis factor-alpha which followed the challenge with LPS or SEB. In sharp contrast, these clinical and seroimmunological events were both markedly counteracted by prophylactic treatment with sodium fusidate, another immunosuppressive agent used as control.

Histopathological studies on antitumor effect of sporamycin. Cell-mediated immunity against allogeneic tumor-bearing mice.

Mice that had received transplants of sarcoma-180 followed by treatment with sporamycin were examined histopathologically at periodic intervals. A marked degeneration of tumor cells was observed at an early stage after the administration of sporamycin, but the degeneration subsequently ceased and regrowth of the tumor was seen. Marked infiltration of lymphoid cells, granulation tissue, and fibrosis was seen in the stroma or surrounding tissue of the tumor at a late stage after the administration of sporamycin, and the regression of tumor cells became marked. With a few exceptions the mice were completely cured by about the 40th day. In the peripheral lymphoid tissues, a transitory decrease in the number of cells was observed after the administration of sporamycin, but this was followed by regeneration of the cells, followed by a marked increase in the B cell system. On the other hand, lymphoid cell depletion of the thymus had persisted. Transplantation of intact sarcoma-180 to mice preliminarily inoculated with sporamycin-treated sarcoma-180 cells resulted in inhibition of tumor growth in most of the mice, and qualitatively the same tissue reactions as those in mice cured of sarcoma-180 by sporamycin were seen. The results suggest that enhancement both of antigenicity of the tumor (cells) and of the subsequent immune response of the host by sporamycin may be involved in the cure of the experimental tumor.

Interferon induction by SMANCS: a polymer-conjugated derivative of neocarzinostatin.

Poly (styrene-co-maleic acid)-conjugated neocarzinostatin, SMANCS, induced antiviral activities in the circulation of mice. The maximum titer of the activity (240 U/ml) was observed 20 h after administration an 8 mg/kg iv dose of SMANCS. Various experiments showed that this antiviral substance induced by SMANCS was an interferon (IFN). Sixty percent of the IFN response stimulated by SMANCS was impaired in mice pretreated with anti-IFN gamma antiserum. This suggests that the serum IFN induced by the agent contained about 60% of IFN gamma. When Thy 1+ or Lyt 2+ T-cells were depleted from mice by administration of anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody (mAb), this IFN response was eliminated. However, when natural killer cells were depleted from mice by treatment with antiasialo GM1 antiserum, no alteration in the IFN response was observed. The SMANCS-stimulated IFN response was also abrogated in mice treated with macrophage blockers (carrageenan and trypan blue), whereas administration of anti-Lyt 1.2 mAb had no effect on the IFN response. These results suggest that macrophage and T-cells w Lyt 1-2+ phenotype may have an important role in the IFN response stimulated by SMANCS.

Monoclonal antibody raised against apoprotein of C-1027: effect on biochemical and biological activities of the holoantibiotic.

A monoclonal antibody raised against C-1027 apoprotein recognized not only the apoprotein, but also the holoantibiotic (Antibiotic C-1027) almost equally. Among the biochemical and biological activities of the holoantibiotic, the antibody inhibited the aminopeptidase activity and the cytotoxicity to Ehrlich carcinoma cells in cultures, but not the DNA-cleaving activity in vitro. The immunohistogram, using this antibody, of Ehrlich carcinoma cells that had been exposed to the holoantibiotic suggested penetration of the holoantibiotic into target cells.