Cryosurgery of the monkey (macaque) prostate. II. Apparent immunopathologic alterations following cryostimulation.

Previous investigation of the development of humoral immunologic responsiveness following cryostimulation of the monkey prostate up to 30 days postoperatively disclosed low to modest titres of antibodies to prostatic tissue antigens. In the present study, the possible occurrence and predominance of a cellular response and its ensuing immunopathologic effects on the prostate and other accessory sexual glands of these animals, not initially examined, together with further serologic evaluation and correlation of this cellular response with the presence of antiprostatic antibodies, has been made 41 to 90 days following freezing. A reduction in the size of the prostate observed following freezing was accompanied by what were suggestive of immunopathologic alterations. Alterations occurred principally in the caudal lobe, and were characterized by what appeared to be specific periacinar foci of lymphocytic infiltrates. These lymphocytes were observed to infiltrate onto the acinar epithelial cells with subsequent separation of epithelial cells from the basal lamina and epithelial cell destruction. Other observed alterations in the prostate consisting of stromal fibrosis, periodic presence of inflammatory cells, proliferation of regenerating glands, and squamous metaplasia were in consonance with previous histologic studies of the prostate following cryosurgery by others. Antiprostatic antibodies were present in only one of the seven animals evaluated at the time which these observations were made. The present preliminary observations provide evidence suggestive of the development of a specific cellular immunologic response following cryosurgery of the prostate. Fending confirmation of the study of a larger series of animals, these observations may be of potential significance in providing an explanation of reported cases of eradication of human prostatic carcinomas following cryotherapy.     

Immunological aspects of cryosurgery in general surgery

Existence and verification of the cryoimmunologic reaction are established in basic experiments and clinical studies. However, the most effective condition in which to elicit cryoimmunologic reaction is still unknown. Evidence suggests the necessity to intensify cryoimmune reaction, if we intend to use this as one of the specific immunotherapies clinically. Establishment of criteria to select patients receiving cryosurgery is another pending question. It is important to assess the patient's immunity or immunological competence prior to cryosurgery, thereby preventing immunologically induced aggravation after cryosurgery.Twelve years of clinical experience in cryosurgery was presented and analyzed. Overall 3-year survival in the primary cancer patients was 33.3%, and 5-year survival was 17.7%. It is our aim to achieve the most beneficial effects of cryosurgery for the patients with malignancies which would otherwise be difficult to treat by conventional means.     

Antitumor immunologic reactivity in the relatively early period after cryosurgery: Experimental studies in the rat

An experimental investigation was performed on antitumor immunity in the relatively early postoperative period after cryosurgery, using a metastasizing rat's mammary tumor, MRMT-1. Two weeks after its inoculation, surgical excision of the tumor, cryosurgery, surgical excision plus inoculation with freezing-thawing produced vaccine, or surgical excision plus fasting for 72 hr was performed, and postoperative follow-up was done on incidences of metastases, those of metastatic death, etc. Specific immunologic reactivity was examined in the surgical excision (SE) and cryosurgery (CR) groups.The FTV and fasting groups showed more metastatic deaths as compared with the SE group. The CR and SE groups did not differ significantly from each other in incidences of lung and lymph node metastases.Specific footpad reactivity at 2 and 3 weeks after treatment was lower in the CR group than in the SE group.Winn's neutralization assay showed an inhibition of tumor growth at 1 and 3 week(s) after treatment both in the SE and in the CR groups, the inhibitory effect tending to be lower in the latter.Inactivated serum obtained at 1 week after treatment showed a facilitation of tumor growth in the SE group and a tendency of tumor suppression in the CR group, showing a significant difference between them.A mild reduction in antitumor immunity seen in the relatively early postoperative period following cryosurgery probably was not due to a blocking effect by superfluous antigens. Rather it was considered to be due to activation of suppressor cells, consequent on cryosurgical stress, and/or slow and steady absorption of antigens.     

Cryosurgery of the rabbit prostate. Comparison of the immune response of immature and mature bucks

The intensity and specificity of the immune response developed following each of three independent in situ freezing insults of the coagulating gland (anterior prostate) of immature male rabbits have been compared to that obtained following similar cryostimulation of the coagulating gland of mature bucks by the methods of tanned cell haemagglutination and gel diffusion precipitation. Results of this study offer confirmation to the previously demonstrated secondary or anamnestic immune response observed following multiple freezing of the rabbit coagulating gland and indicate further that the size and physiological status of the coagulating gland, i.e., the concentration of coagulating gland secretory autoantigens, are among the important variables in determining the cryosensitivity of a given animal. This cryosensitivity may be of particular significance in the prospective treatment of patients with prostatic cancer by cryoimmunotherapy. That is, the development of an immune response in such patients may reasonably be related to the concentration and subsequent release of prostatic tumourspecific or tumour-associated antigens.     

Late appearance of resistance to tumor rechallenge following cryosurgery: A study in an experimental mammary tumor of the rat

Resistance to tumor challenge following surgical and cryosurgical eradication of the tumor was studied, using an experimental mammary tumor of the rat, MRMT-1. It was revealed that rejection rate of the challenged tumor increased gradually following cryosurgery and reached its peak at 10 weeks after cryosurgery. No such phenomenon was observed after surgical excision of the tumor. Decreased incidence of lymph node metastases and decreased tumor weights in “take” cases also suggested an increased immunological activity against the tumor at 10 weeks after cryosurgery.     

Cryoimmunotherapy with local co-administration of ex vivo generated dendritic cells and CpG-ODN immune adjuvant,elicits a specific antitumor immunity

Cryoablation is a low-invasive surgical procedure for management of malignant tumors. Tissue destruction is obtained by repeated deep freezing and thawing and results in coagulative necrosis and in apoptosis. This procedure induces the release of tumor-associated antigens and proinflammatory factors into the microenvironment. Local administration of immature dendritic cells (DCs) potentiates the immune response induced by cryoablation. To further augment the induction of long-lasting antitumor immunity, we investigated the clinical value of combining cryoimmunotherapy consisting of cryoablation and inoculation of immature DCs with administration of the immune adjuvant, CpG oligodeoxynucleotides. Injection of the murine Lewis lung carcinoma, D122-luc-5.5 that expresses the luciferase gene, results in spontaneous metastases, which can be easily monitored in vivo. The local tumor was treated by the combined treatment. The clinical outcome was assessed by monitoring tumor growth, metastasis in distant organs, overall survival, and protection from tumor recurrence. The nature of the induced T cell responses was analyzed. Combined cryoimmunotherapy results in reduced tumor growth, low metastasis and significantly prolonged survival. Moreover, this treatment induces antitumor memory that protected mice from rechallenge. The underlying suggested mechanisms are the generation of tumor-specific type 1 T cell responses, subsequent induction of cytotoxic T lymphocytes, and generation of systemic memory. Our data highlight the combined cryoimmunotherapy as a novel antitumor vaccine with promising preclinical results. Adjustment of this technique into practice will provide the therapeutic benefits of both, ablation of the primary tumor and induction of robust antitumor and antimetastatic immunity.     

Cyclophosphamide pretreatment in tumor cryotherapy: A comparison with alternative drugs

The method of Turk and Lagrange for modulating immune responses in favor of the cell-mediated effector arm by using single high doses of cyclophosphamide 3 days before antigen has previously been shown to cause decreased rates of local tumor recurrence when adapted as an adjunctive therapy to cryosurgery in a murine model. This series of experiments compares cyclophosphamide, azathioprine, and methotrexate against cryosurgery alone in the same therapeutic protocol. Only cyclophosphamide gave enhanced numbers of cures; azathioprine caused an increase in metastases arising concurrently with local tumor recrudescence.     

Immunocompetence and helminth community of the white-toothed shrew, <Emphasis Type="Italic">Crocidura russula</Emphasis> from the Montseny Natural Park,Spain

Host immunocompetence assessed by spleen size and response to phytohaemagglutinin (PHA) injection may give some indications on the control of parasite infection and on host mediation effect, through immunity, on parasite community structure. We investigated the helminth community and immunocompetence of the white-toothed shrew in a small area to test the relationship between immunocompetence and intensity of helminth infection. At the proximate level and if spleen mass and PHA response reflect the level of immunocompetence, we expected that individuals with a large spleen or a high PHA response should harbour a lower parasite load than individuals with relatively small spleen or low PHA response. In addition, we predicted that the structure of the helminth community should be mediated by the host’s immune defence. Spleen mass was linked to helminth infection. Nematodes and cestodes were negatively associated within hosts. PHA response was not related to total helminth intensity of infection but was negatively related to cestode intensity and positively to nematode intensity. This result suggests either a differential modulating effect on immunity by the two groups of worms or the existence of an antagonistic association between nematodes and cestodes mediated by the immune response of the host.     

Route of Administration of the TLR9 Agonist CpG Critically Determines the Efficacy of Cancer Immunotherapy in Mice

Background

The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ.

Methodology/Principal Findings

In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone.

Conclusions/Significance

CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting.     

Investigation of the mechanism and the effect of cryoimmunology in the Copenhagen rat

This study examined the potential for "cryoimmunology" to increase the destruction of the Dunning AT-1 prostate tumor after cryosurgery. Two possible mechanisms explaining the cryoimmunologic response were studied. The first was that an antitumor antibody is produced after cryosurgery. The second was that freezing induces an immunostimulatory signal that creates a T-cell response to the tumor. Six groups of animals (three experimental groups and three control groups) were treated once per week for 4 weeks with different therapies designed to investigate these mechanisms. Three types of immune response were measured: (1) the anti-AT-1 tumor immune titer (Ab response) by serum ELISA, (2) the effect on secondary tumor growth after challenge with live AT-1 cells (size and weight of the secondary tumor over time), and (3) the nature of the immunologic infiltrate into the secondary tumors by immunoperoxidase stain. ELISA showed that immune titers were present in the experimental groups after therapy, but the presence of an immune titer did not have a significant effect on tumor propagation. Histology showed the immunologic infiltrate was similar in all groups. These results showed that an immune response to AT-1 tumor was measurable by serum antibody, but it did not significantly limit secondary tumor growth or affect tumor histology. This suggests that the growth of AT-1 tumors is not inhibited by a cryoimmunological response. Thus, the effect of in vivo cryosurgery in the AT-1 tumor system would likely be limited to cellular and vascular changes.     

Cyclophosphamide pretreatment in tumor cryotherapy: A murine model

Following the protocol of Turk and Lagrange for selective potentiation of cell-mediated immunity, a single high dose of cyclophosphamide was given to inbred mice bearing subcutaneous tumors 3 days before cryosurgery. Cyclophosphamide pretreatment caused a rise in the rate of total tumor ablation and local recurrence was delayed. The dose response to tumor size, cyclophosphamide, and freeze parameters indicated that these effects were the result of synergistic rather than additive processes. Instances of metastatic tumor growth and immunologically mediated complications of cryosurgery were also lower when the cryosurgery was accompanied by cyclophosphamide pretreatment.     

Bill colour and immunocompetence in the European blackbird

The level of expression of secondary sexual characters has been suggested to signal male ability to resist parasitic infestations. To test this idea, several studies have examined the link between sexual signals and immunocompetence in birds. However, most of them have used only a single aspect of immune response to evaluate immunocompetence. We investigated the relation between bill colour and immunocompetence in captive male European blackbirds, Turdus merula, during the breeding season by assessing both cell-mediated and humoral components of the immune system. The blackbird is a sexually dimorphic species with bill colour varying from yellow to orange in males. Humoral immunity was assessed by measuring both primary and secondary responses to sheep red blood cell inoculation. Cell-mediated immunity was estimated with a delayed cutaneous hypersensitivity response to an injection of a mitogen (phytohaemagglutinin). No relation was found between male bill colour and the primary humoral response. However, males with orange bills showed a lower secondary humoral response but a higher cell-mediated immune response than males with yellow bills. Thus, the relation between immunocompetence and a secondary sexual trait may differ markedly depending on which component of the immune system is under consideration. We discuss our results in relation to mechanisms involved in sexual selection. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour      

Head Exposure to Cold during Whole-Body Cryostimulation: Influence on Thermal Response and Autonomic Modulation

Recent research on whole-body cryotherapy has hypothesized a major responsibility of head cooling in the physiological changes classically reported after a cryostimulation session. The aim of this experiment was to verify this hypothesis by studying the influence of exposing the head to cold during whole-body cryostimulation sessions, on the thermal response and the autonomic nervous system (ANS). Over five consecutive days, two groups of 10 participants performed one whole-body cryostimulation session daily, in one of two different systems; one exposing the whole-body to cold (whole-body cryostimulation, WBC), and the other exposing the whole-body except the head (partial-body cryostimulation, PBC).10 participants constituted a control group (CON) not receiving any cryostimulation. In order to isolate the head-cooling effect on recorded variables, it was ensured that the WBC and PBC systems induced the same decrease in skin temperature for all body regions (mean decrease over the 5 exposures: -8.6°C±1.3°C and -8.3±0.7°C for WBC and PBC, respectively), which persisted up to 20-min after the sessions (P20). The WBC sessions caused an almost certain decrease in tympanic temperature from Pre to P20 (-0.28 ±0.11°C), while it only decreased at P20 (-0.14±0.05°C) after PBC sessions. Heart rate almost certainly decreased after PBC (-8.6%) and WBC (-12.3%) sessions. Resting vagal-related heart rate variability indices (the root-mean square difference of successive normal R-R intervals, RMSSD, and high frequency band, HF) were very likely to almost certainly increased after PBC (RMSSD:+49.1%, HF: +123.3%) and WBC (RMSSD: +38.8%, HF:+70.3%). Plasma norepinephrine concentration was likely increased in similar proportions after PBC and WBC, but only after the first session. Both cryostimulation techniques stimulated the ANS with a predominance of parasympathetic tone activation from the first to the fifth session and in slightly greater proportion with WBC than PBC. The main result of this study indicates that the head exposure to cold during whole-body cryostimulation may not be the main factor responsible for the effects of cryostimulation on the ANS.     

Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian

Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus—infection load, survival rate, and host immunocompetence—following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95 days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation.     

Histologic study of recurrent basal cell carcinoma

This retrospective study of recurrent basal cell carcinoma suggests the presence of irregularity in the peripheral palisade (IPP), squamous differentiation (SD), and tumor ulceration as histologic criteria for the recurrent tumor and the presence of infiltrates of small lymphocytes (LI) in the tumor as a criterion of tumor-directed host immunocompetence. The presence of either irregularity in the peripheral palisade in greater than 75 percent of the tumor cords or absence of infiltrates of small lymphocytes at the tumor base were each significant at the p less than 0.001 level in characterizing recurrent basal cell carcinoma.     

Immune Phenotype and Body Condition in Roe Deer: Individuals with High Body Condition Have Different,Not Stronger Immunity

An efficient immunity is necessary for host survival, but entails energetic costs. When energy is limited, immunocompetence and body condition should co-vary positively among individuals and, depending on body condition, individuals should allocate more either in innate immunity or in adaptive response. We tested whether immune phenotype depends on body condition in large mammals, using data from two contrasted populations of roe deer Capreolus capreolus in France. Roe deer living at Chizé, a forest with poor habitat quality, were expected to show lower values for body condition and immune parameters than roe deer at Trois Fontaines, a forest with high habitat quality. From 285 blood samples collected between December 2009 and March 2011, we measured seven metabolic parameters and ten immunological parameters. A Principal Component Analysis showed that all indicators of body condition co-varied positively and were lowest at Chizé. Several immunological indicators correlated to body condition and differed between Trois Fontaines and Chizé. However, high body condition was not associated to a high average level of immunocompetence, but instead to high levels of indicators of acute inflammatory innate response, while low body condition was associated to high levels of monocytes and lymphocytes, possibly reflecting adaptive immunity. Limited data suggest that the difference between populations was not related to the presence of specific parasite species, however parasite exposure and stress have to be investigated to gain a more complete understanding of the determinants of immunity.     

Cryosurgery of pulmonary metastases.

Indications and results of 33 cryosurgical interventions for metastatic tumors in the lung are presented. Regression of local and metastatic pulmonary growth on the contralateral side was observed in four cases. Nine cases showed temporary halt of metastatic pulmonary tumor growth. The technique of cryosurgery for pulmonary metastases is reviewed. The procedure of cryosurgery of pulmonary metastases was found to be an innocuous method to attempt both tumor destruction and eventually specific immunologic stimulation. Preliminary observations with the lymphocytes and sera indicate that cryosurgery of pulmonary metastases induces an increase in specific cell mediated immune response without producing blocking serum factors and may give rise to specific, complement dependent cytotoxic antibodies. In one case both mechanisms were observed after cryotherapy. In three cases with progressive disease, lymphocyte mediated cytotoxicity alone was stimulated.     

Immunomodulatory Agents as Adjunctive Therapy for the Treatment of Resistant Candida Species

Invasive Candida infections have increased fivefold over the past 20 years. During this time, the incidence of antifungal resistance and infection due to non-albicans species has risen with the increasing use of broad spectrum antifungals. As few new antifungal agents are in development, strategies to improve outcomes in the treatment of Candida infections are sorely needed. The use of immunotherapy to augment the host immune response as an adjunctive treatment for Candida infections is a potentially robust and promising approach. The purpose of this review is to focus on new developments in the use of adjunctive immunotherapy for the treatment of Candida infections, and discuss the potential impact of antifungal resistance on the host immune response.     

Lenalidomide enhances anti-myeloma cellular immunity

Lenalidomide is an effective therapeutic agent for multiple myeloma that exhibits immunomodulatory properties including the activation of T and NK cells. The use of lenalidomide to reverse tumor-mediated immune suppression and amplify myeloma-specific immunity is currently being explored. In the present study, we examined the effect of lenalidomide on T-cell activation and its ability to amplify responses to a dendritic cell-based myeloma vaccine. We demonstrate that exposure to lenalidomide in the context of T-cell expansion with direct ligation of CD3/CD28 complex results in polarization toward a Th1 phenotype characterized by increased IFN-γ, but not IL-10 expression. In vitro exposure to lenalidomide resulted in decreased levels of regulatory T cells and a decrease in T-cell expression of the inhibitory marker, PD-1. Lenalidomide also enhanced T-cell proliferative responses to allogeneic DCs. Most significantly, lenalidomide treatment potentiated responses to the dendritic cell/myeloma fusion vaccine, which were characterized by increased production of inflammatory cytokines and increased cytotoxic lymphocyte-mediated lysis of autologous myeloma targets. These findings indicate that lenalidomide enhances the immunologic milieu in patients with myeloma by promoting T-cell proliferation and suppressing inhibitory factors, and thereby augmenting responses to a myeloma-specific tumor vaccine.