Endometriosis prophylaxis and treatment with the newly developed xenogenic immunomodulator RESAN in an animal model.

OBJECTIVE: The objective was to assess the effectiveness of the newly developed immunomodulator RESAN in the prophylaxis and treatment of endometriosis induced in rats. STUDY DESIGN: The study was performed on 58 Wistar rats. Twelve weeks before endometriosis induction, the RESAN vaccine was administered to 24 rats (100 mg i.m. and 100 mg s.c.). Endometriosis induction was performed in 48 rats, which were divided into two groups: group I, the prophylaxis group, consisting of 24 previously vaccinated rats; and group II, the treatment group, comprising the other 24 rats, which had not been vaccinated. The graft (4 mm x 4 mm) of endometrium was attached to the parietal peritoneum. A sham operation was performed in 10 rats (group III). After 3 months, a second laparotomy was performed in all animals, and endometriotic foci were excised when present. RESAN was administered to the group II animals. After an additional 3 months, a third laparotomy was performed in all animals of the three groups. RESULTS: Positive, histologically confirmed endometriosis was found in 4.3% of the animals in group I and in 69.6% of group II rats (p<0.0001). Macroscopic assessment revealed endometriosis in 21.7% and 91.3% of animals in groups I and II, respectively (p<0.0001). At final laparotomy, 3 months after excision of the previously suspected foci, no signs of endometriosis were found according to both macroscopic assessment and histological examination. During the second laparotomy intraperitoneal adhesions were present in 13.0% of the animals in group I and in 61.0% of those in group II. No adhesions were present in group III. At the final laparotomy, the adhesions were present in only three of the animals in group II (p<0.0009). CONCLUSIONS: RESAN seems to be effective in both the prophylaxis and treatment of endometriosis, as well as in the prophylaxis of adhesions. Histological confirmation of endometriosis should be mandatory.

A new strategy to induce effective antitumour response in vitro and in vivo.

To induce Her2-specific cell immune response, we used xenogeneic antigen rat neu L2-S2 domains as the vaccine antigen. The antigenic protein was engineered as a chimeric protein with human IgG1 Fc region (neu-Fc). Neu-Fc could stimulate the cell proliferation in mixed lymphocyte reaction effectively. Simultaneous neu-Fc and IFN-gamma stimulation dramatically elevated IL-12 secretion and reduced IL-10 production in PBMC. To further augment the activating effects on Th1-type response, Bacille Calmette-Guerin (BCG) was utilized as a non-specific stimulus. Neu-Fc, IFN-gamma and BCG costimulation exhibited the most conspicuous effects on the reversal of the Th1-type inhibitory effects by MCF-7 cell supernatant compared with neu-Fc alone or IFN-gamma and BCG costimulation. The lytic activity of effector cells to Her2 overexpressing cells was greatly promoted by neu-Fc, IFN-gamma and BCG stimulation simultaneously. Neu-Fc led to considerable retardation in EMT6/Her2 tumour growth in Balb/c mice. IFN-gamma and BCG efficiently enhanced the antitumour activity. A large amount of inflammatory cells were found to be accumulated in the tumour tissues or surrounded tumours in mice treated with neu-Fc, IFN-gamma and BCG but no inflammatory cell infiltration was observed in control tumours, indicating that the strategy is potent enough to support the initiation and propagation of tumour-specific immune response in an established tumour and generate a proinflammatory environment.

Combination of low-dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities.

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low-dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low-dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody-producing B cells against mouse self-endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low-dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.

DNA vaccines: an active immunization strategy for prostate cancer.

Immunotherapy is currently being investigated as a treatment for patients with asymptomatic, recurrent prostate cancer manifested only by a rising prostate-specific antigen (PSA) level. Several different approaches to active immunization against antigens found on cancer cells have been explored. Immunization with DNA overcomes many of the obstacles noted in previous studies. Injection of plasmid DNA encoding a xenogeneic differentiation antigen (prostate-specific membrane antigen [PSMA]) is a potent means to induce antibody and T-cell responses to these otherwise poorly immunogenic self proteins. Use of the xenogeneic DNA (ie, human PSMA DNA injected into mouse) has been shown to be an absolute requirement to overcome immunologic tolerance. We are currently conducting a phase I trial of human and mouse PSMA DNA vaccines in patients with recurrent prostate cancer, based on preclinical experiments described below.

Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor.

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4(+) T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8(+) cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8(+)) or CD8-depleted (CD4(+)) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-gamma and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.

Induction of complement attack on human cells by Gal(alpha1,3)Gal xenoantigen expression as a gene therapy approach to cancer.

Galactose(alpha1,3)galactose on the surface of cells of non-primate organs is the major xenoantigen responsible for hyperacute rejection in xenotransplantation. The antigen is synthesised by (alpha1, 3)galactosyl transferase. Humans lack this enzyme and their serum contains high levels of pre-existing natural antibody which recognises the structure and activates complement. We have evaluated in vitro the potential for delivery of this enzyme to sensitise human cells to complement attack as a gene therapy approach to cancer. Retrovirus-mediated delivery of (alpha1,3)galactosyl transferase resulted in high level expression which led to serum-mediated lysis of five human cell targets, including endothelial and primary melanoma cells. Lysis was specific for those cells expressing the antigen in a mixed cell population. The mechanism of cell lysis mimicked that involved in hyperacute rejection: activation of the classical complement pathway by natural antibody specific for galactose(alpha1,3)galactose. The degree of lysis was determined by both the level of specific antibody and the expression of glycophosphatidylinositol-linked complement regulatory proteins. We conclude that expression of (alpha1,3)galactosyl transferase is a promising new therapeutic approach for cancer gene therapy, avoiding toxicity problems associated with application of prodrugs and with the potential to elicit further immunological responses.

Evaluation of intradermal sensitivity test in patients submitted to heterologous serotherapy

This review deals with heterologous sera produced used in Brazil. The authors studied 64 patients. Of these, 35 had been attacked by domestic and wild animals and received antirabies serum; 20 had been bitten by venomous animals (snakes and scorpions) and were treated with specific antivenoms; and 9 had traumas and received antitetanic serum. All these patients were submitted to the intradermal sensitivity test before, and 30 days after receiving heterologous serotherapy. The following results obtained by the authors agree with those in literature: - The intradermal test using undiluted heterologous serum produced a more acute reaction than that using heterologous serum diluted 1:10; - The larger the volume of serum, the larger was the wheal directly after inoculation; - The antigenic concentration influenced the final local reaction; - The reading 30 minutes after inoculation was always higher than that 15 min after; - No systemic reaction was observed during the tests; - The use of promethazine as a prophylactic medication did not protect against the early reactions; - Tests performed 30 days after serotherapy showed a higher reactivity, probably due to sensitization; - The intradermal sensitivity test did not allow the authors to predict early reactions. After these observations, the authors do not recommend the intradermal sensitivity test for patients submitted to heterologous serotherapy. They, however, strongly recommend a careful observation during the infusion and clinical follow up in the first 24 hours.