Influence of poly A-poly U on early events in the immune response in vitro

Adjuvant action of artificial homopolyribonucleotides and nucleic acid digests was studied in mouse spleen-cell cultures, stimulated with sheep red cells by the technique of Mishell and Dutton. Assays of antibody-forming cells were performed according to the Jerne and Nordin technique. Doublestranded poly A-poly U, and RNA digests had adjuvant activity over a wide dose range (0.01–100 μg/ml). DNA digests, single stranded polynucleotides (poly A, poly U, poly C) and mixtures of ribonucleosides and ribonucleotides had little or no enhancing effect at comparable doses. From our study of the kinetics of increase of plaque-forming cells in cultures treated with poly A-poly U, in conjunction with in vivo studies of others, we concluded that adjuvant action, during induction, indirectly increased the number of bone marrow-derived cells that responded to antigenic stimulation by increasing the effectiveness of the thymus-derived cells in the culture.     

Study of the hydratation of the double-helical poly A-poly U complex by IR-spectroscopy and piezogravimetry methods

IR-spectra of a double-helical poly A-poly U complex and coiled poly U were studied at various r.h. in a 900-3800 cm-1 region. By the method of piezomicrobalance hydration isotherms for these polynucleotides were obtained. It is concluded that, as in the DNA case, simultaneous hydration of nucleic bases the backbone of polynucleotides occurs at lower r.h., and that the poly A-poly U hydration level is higher than poly A and poly U ones separately. Drastic changes in spectral parameters of poly A poly U uracil and adenine in-ring and out-of-ring absorption bands observed in 44-76% r.h. region were interpreted as a transition to helical conformation of the complex. Calculation of resonance frequencies for these normal vibrations in the dipole-dipole approximation agrees with the experimental data.     

Naringenin reduces lung metastasis in a breast cancer resection model

Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.     

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.     

Perioperative blood transfusion is associated with poorer survival in patients with gastric cancer

The putative prognostic significance of perioperative blood transfusions on gastric cancers is controversial and the published results are contradictory. The aim of this study was to evaluate the prognostic influence of transfusion on Chinese gastric cancer surgery. Six hundreds and seventy-six patients who underwent curative gastrectomy for gastric cancer from 2000 to 2004 were retrospectively reviewed. Uni- and multivariate analyses of the incidence and amount of transfusion, and a comparison of the clinicopathological features were performed. Subgroup analyses of prognosis according to stage, tumor size, and pretreatment anemia were carried out. Blood transfusion was significantly associated with older age (>60 year), larger tumor (>6 cm), upper and middle location, surgical margin status, and pretreatment anemia. In addition, tumors in the transfused group were more advanced in depth of invasion, nodal stage, and TNM stage. No significant relationship was found between the amount of transfused blood and prognosis. Subgroup analyses of prognosis according to stage showed significant differences in stages II and III, between the transfused and nontransfused groups. Significant difference between the transfused and nontransfused groups could be observed in two subgroups of tumor size. Patients with or without anemia in the nontransfused group both had a longer survival time than those in the transfused group. On multivariate analysis, transfusion was shown to be an independent risk factor for poor prognosis. This study suggests that perioperative blood transfusion is associated with a significantly worse prognosis following gastric cancer surgery. The parameters such as advanced stage, tumor size, and anemia do not affect its prognostic value.     

Sensitized fluorescence in polynucleotide-dye complexes and the problem of energy transfer in polynucleotides

New results on sensitization of fluorescence of Profiuvine, Acridine Orange and Ethidium Bromide complexes wilh double stranded poly A-poly U upon ultraviolet irradiation are presented. Together with older results on dye DNA complex, they are interpreted to show that base to base transfer plays essentially no role compared to direct base to dye transfer. A lower limit of base to base transfer time (which is at the same time a higher limit for life time of fluorescence) in DNA (2. 10^?11 sec.) and poly A-poly U (2. 10^?13 sec.) is obtained which cast some doubt upon the validity of calculations of optical properties using the exciton (strong coupling) approximation.     

Macrophage prostaglandin E2 and oxidative responses to endotoxin during immunosuppression associated with anaesthesia and transfusion

The widespread use of blood transfusion in major surgical procedures has led to concern about the immunosuppressive effect of transfusion on patients with underlying malignancy. Transfusion may also suppress the host response to infection. The cellular mechanisms of transfusion-associated immunosuppression may involve macrophage prostaglandin E₂ (PGE₂) in modulating the host response to cancer and infection. We previously observed that the transfusion of blood increased PGE₂ production by unstimulated macrophages. To investigate this PGE₂ associated immunosuppression, we studied the effect of transfusion of rats using a physiological stimulus of macrophage PGE₂ production, bacterial endotoxin. In the same macrophages, we analysed intracellular oxidative activity. Both allogeneic and syngeneic blood transfusion were associated with increased PGE₂ release by macrophages. This stimulation of PGE₂ increased with duration of storage of blood. A similar effect of serum indicated that a humoral factor was involved. Endotoxin (50 ng/ml–500 μg/ml) stimulated PGE₂ production in all transfused subjects. The lowest endotoxin concentration gave proportionately the greatest stimulation. Oxidative activity was down-regulated in macrophages of transfused rats, supporting an immunosuppressive role of PGE₂ within the macrophage. An effect of surgery on the oxidative response was also detected.     

Recombinant human erythropoietin corrects anemia of blood loss: a study in the dog

In order to evaluate the possibility of using recombinant human erythropoietin (rhEpo) for the prevention and correction of anemia due to blood loss and as an adjuvant for autologous blood transfusion, its preventive and therapeutic effects were evaluated in beagles in which anemia was induced by repeated phlebotomies. Two hundred U/kg of rhEpo were administered i.v. four or nine times every two weeks for six weeks. Phlebotomies (25 ml/kg of body weight) were conducted three times at two-week intervals. rhEpo was found to successfully prevent and correct anemia caused by the phlebotomies. Concurrent administration of iron increased efficacy. The findings obtained in the present study suggest that rhEpo is useful both for the treatment of anemia caused by blood loss due to surgery and as an adjuvant therapy for pre-deposit autologous blood transfusion.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Effects of intravascular laser irradiation of blood on growth and metastasis of lymphosarcoma in rats

The effect of intravenous blood radiation by low-intensity laser on tumour growth in Wistar rats with Pliss lymphosarcoma has been studied. As a result of single or double blood radiation by laser with wave-length of 510 and 633 nm both the tumour growth inhibition and metastases disappearance have been shown.     

Present status of BCG immunotherapy of malignant melanoma

Summary BCG systemic adjuvant immunotherapy may be effective for improving both the recurrence and survival rates in patients with regional metastases from malignant melanoma. Clinical trials show that many of the principles derived from the study of animal tumor systems are applicable to human cancer in that immunotherapy is most effective for a small residual number of tumor cells. BCG treatment fulfills many of the ideal criteria for adjuvant treatment following surgery when disease burden is lowest. It is relatively nontoxic; it is effective for disseminated melanoma; it has systemic activity in the adjuvant treatment of subclinical metastases. However, until clinical trials are complete, BCG adjuvant therapy must be considered investigational.Supported by USPHS grants CA05252, CA12582, and NIH 0732001 CB43852.     

Detection and significance of minimal residual disease in colorectal cancer

Colorectal cancer (CRC) is one of the most common causes of cancer death in the developed world. Although the primary treatment for CRC is surgical, disease relapse due to minimal residual disease (MRD) following apparently curative surgery occurs in up to fifty percent of patients. Most patients who develop overt metastases beyond the regional lymph nodes eventually die of the disease. At present adjuvant chemotherapy is used to improve survival in patients with metastases to regional lymph nodes demonstrated by routine histopathology with no other evidence of spread. The ability to identify metastatic disease at an earlier stage could be of considerable benefit in directing adjuvant therapy to patients at high risk of relapse who are not identified by current methods. Several techniques have been developed for the detection of MRD, including immunohistochemical and molecular methods, however their role in clinical practise is not yet established. The purpose of this paper is to review these techniques and their potential clinical use in the management of CRC.     

Targeting the primary tumor to generate CTL for the effective eradication of spontaneous metastases

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104L(d) that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.     

VEGF levels and the angiogenic potential of the microenvironment can affect surgical strategy for colorectal liver metastasis

The hypotheses emerging from basic research on colorectal liver metastases must be tested in clinical situations for the adaptation of current treatment strategies. Pre-metastatic niches have been shown to exist in human colorectal synchronous metastases, with the liver parenchyma adjacent to the synchronous liver metastases providing a favorable, angiogenic environment for metastatic tumor growth. The role of the VEGF signaling pathway in liver regeneration and tumor growth remains unclear, but the use of antiangiogenic agents in combination with surgical treatment is almost certainly beneficial.     

VEGF levels and the angiogenic potential of the microenvironment can affect surgical strategy for colorectal liver metastasis

The hypotheses emerging from basic research on colorectal liver metastases must be tested in clinical situations for the adaptation of current treatment strategies. Pre-metastatic niches have been shown to exist in human colorectal synchronous metastases, with the liver parenchyma adjacent to the synchronous liver metastases providing a favorable, angiogenic environment for metastatic tumor growth. The role of the VEGF signaling pathway in liver regeneration and tumor growth remains unclear, but the use of antiangiogenic agents in combination with surgical treatment is almost certainly beneficial.     

Adjuvant immunotherapy for solid tumors: from promise to clinical application

Although surgery remains the mainstay for the treatment of most solid tumors, investigators are seeking complementary therapies to eradicate microscopic disease, which causes tumor relapse even after an apparently complete surgical excision. Although adjuvant chemotherapy has achieved some significant results, the control of minimal residual disease is still a challenge for clinicians. Among novel therapeutic approaches, immunotherapy holds promise. This anticancer strategy aims at triggering a highly specific endogenous killing machine against tumor cells. Recent progress in tumor immunology has improved our understanding of host-immune system interactions. In particular, new technologies have fostered the identification of potentially immunogenic tumor antigens that can be used as suitable targets for immune effector cells. After observing immunotherapy-mediated clinical responses in patients with metastatic disease, investigators have started evaluating this anticancer modality in the adjuvant setting. Here, we review the immunological strategies so far explored in humans and report worldwide results following the clinical application of adjuvant immunotherapy for solid tumors.     

Immune reconstitution prevents metastatic recurrence of murine osteosarcoma

Primary tumors developing in immunocompetent hosts escape immunosurveillance by acquiring immune evasive properties. This raises the prospect that metastases derived from such tumors will also evade immunity. To investigate whether immune surveillance plays a role in preventing metastases, we studied a murine model which mimics the clinical progression of osteosarcoma: primary tumor growth in the lower extremity, amputation, minimal residual disease followed by the development of overt metastases. K7M2 implants readily escaped immune surveillance since normal BALB/c mice, T cell deficient SCID and T/NK cell deficient SCID-bg mice showed no difference in the rate of growth of primary osteosarcomas. However, both SCID and SCID-bg mice had higher rates of metastases than immunocompetent mice. Similarly, immune reconstitution following transfer of naive T cells to SCID or SCID-bg mice did not impact primary tumor growth, but significantly diminished metastatic recurrence. T cells in osteosarcoma bearing mice produced IFNγ in response to tumor and IFNγ production by immune reconstituting T cells was required to prevent metastases. These results demonstrate an important role for T cell based immune surveillance in preventing metastases, even when metastases develop from tumors that adeptly evade immunosurveillance. The results further suggest that T cell depleting cancer therapies may eliminate beneficial immune responses and that immune reconstitution of lymphopenic cancer patients could prevent metastatic recurrence of solid tumors. By acceptance of this article, the publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Animal care was provided in accordance with procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH Pub. No. 86-23, 1996). This project was funded in whole or part with funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.     

Mechanism of adjuvant activity of poly A:U on antibody responses to hapten-carrier conjugates

The adjuvant action of poly A:U has been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. Administration of poly A:U at the time of primary immunization with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) shortens the induction period for, and heightens the magnitude of peak anti-DNP antibody and specific memory cell production. In order to define the cellular locus of poly A:U action, the effect of this adjuvant on adoptive secondary anti-DNP antibody responses was studied. Spleen cells from DNP-KLH-primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine gamma globulin (BGG), can be stimulated to do so when an appropriate dose of poly A:U is administered with DNP-BGG. The capacity for poly A:U to exert this effect requires the presence of T lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro prior to adoptive transfer abrogates the response to DNP-BGG plus poly A:U. Moreover, evidence is presented that demonstrates that poly A:U exerts its adjuvant action on the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of poly A:U to augment adoptive secondary anti-DNP responses to the DNP derivative of a nonimmunogenic copolymer of d-glutamic acid and d-lysine (d-GL) for which there are few or no specific, functional T cells.     

The immunomodulation effect of allogenic blood transfusion in colorectal cancer--a new approach

The total number of 542 patients with colorectal cancer surgery have been analyzed in order to estimate the effect of receiving transfusion local recurrences, and the disease free - survival. It should be examined whether there are changes in general immunity indicators which would be connected with perioperative transfusion. A significant connection has been found between local recurrences and blood transfusion (p<0.0001), the most noticeable being in Dukes A (p =0.045), localization on rectum (p=0.036). The receiving of blood transfusion is linked significantly with disease free - survival reduction (p =0.0068; log rank), the most significant being in Dukes A stage (p =0.0123; log rank) and with localization on rectum (p=0.0231). The analysis of general immunity indicators has shown significant immunocompromitation of patients just before the surgery and this could have effect on immunomodulation caused by transfusion and just as on the treatment prognosis of colorectal carcinoma.     

Blood salvage, proper choice for tumor patients?

Blood transfusion is frequently used in tumor patients, However, allogeneic blood transfusion have been claimed to be associated with an increased risk of negative outcomes, including transfusion reactions, fever, virus transmission, and alloimmunization. Other risks of homologous transfusion specific to the tumor patients are the potential deleterious effects on the recurrence of tumor and indefinite overall survival. On the contrary, autologous blood transfusion offers survival advantages to tumor patients and has been shown to minimize the occurrence of many detrimental allogeneic blood associated effects and complications. It also reduces the volume of banked blood needed and improves the prognosis of patients. However, the quality of salvaged blood is still a matter of debate, because it may contain tumor cells which are associated with potential detrimental effects such as metastasis. So, an advanced technology is needed to remove such contaminants to make autologous blood transfusion safer.