Immunotoxicity of medical devices. Symposium overview.

Determination of the ability of a medical device to interact with the immune system currently involves assessment of the immunogenic potential and biocompatibility of the device or an extract of the device. However, implants are often in the body for extended periods of time and/or are placed by a surgical procedure that in and of itself will generate an acute inflammatory response. This symposium discussed studies that have been performed to evaluate the immunogenicity of various devices consisting of several different compositions (i.e., silicone, metals, and latex) in contact with different anatomical sites, the ability of a device to modulate an inflammatory response generated by a surgical procedure or trauma, and the response of the body to a material left in place for extended periods of time. This symposium brought together scientists from many different disciplines to begin to identify and fill in the gaps in this area.

Characterization of the allergen(s) in latex protein extracts.

BACKGROUND: Immediate hypersensitivity to latex, induced by natural latex proteins remaining on the finished products, may lead to severe anaphylactic reactions. METHODS: We investigated the distribution of latex proteins by molecular weight and identified the specific allergenic molecules. Proteins extracted from various latex products were compared with those extracted from raw latex sap, both ammoniated and nonammoniated. RESULTS: Variations in the levels of extractable protein, as well as in the number of molecules and the molecular weight distribution, were observed especially among finished latex products. To identify allergenic (i.e., IgE-binding) molecules, we performed immunoblots with the sera from latex-sensitive persons. The results indicated that antigenic molecule profiles differed among the products and also between the finished products and the raw material. In addition, specificities of the anti-latex IgE antibodies varied among the sensitized persons. CONCLUSIONS: It appeared that persons with the same history of sensitization had similar patterns of antigenic specificities. If the history of exposure, as well as genetic predisposition and medical history of the patient, plays a significant role in the specific IgE response, it may be difficult to select a "standard" antigen and a "standard" antiserum for the evaluation of the latex sensitivity and allergenicity.

Cornstarch powder on latex products is an allergen carrier.

Allergic reactions of the upper respiratory tract during use of powdered latex rubber gloves have been recently associated with sensitivity to latex. We have studied the ability of cornstarch powder to bind latex proteins and evaluated allergenic properties of the bound protein. Allergenicity was determined by competitive inhibition of human anti-latex IgE binding to solid-phase latex antigen. Cornstarch extracted from powdered latex products and clean cornstarch exposed to latex protein extracts were evaluated in comparison with clean unexposed cornstarch. Both exposed cornstarch preparations inhibited specific binding of anti-latex IgE antibodies to latex proteins in a dose-response manner. Latex-exposed cornstarch diluted 50% vol/vol produced complete inhibition, whereas greater dilutions exhibited variable levels of inhibition, depending on the source of cornstarch-bound proteins, insolubilized latex proteins, and IgE antibody-containing human serum used. Cornstarch not exposed to latex had no inhibitory activity. The study demonstrates that cornstarch indeed binds allergenic latex proteins and supports the causative relationship between allergic reactions in individuals with latex sensitivity and the exposure to airborne particles from powdered latex products.

Latex-associated allergies and anaphylactic reactions.

The recent reports of severe anaphylactic reactions and several fatalities caused by contact with latex-containing products raised concerns in the medical community. Although hypersensitivity to natural rubber has been widely reported in the literature, the prevalence and severity of reactions have rapidly increased in the last few years. Latex proteins, constituents of natural latex, appear to be responsible for the sensitization. Many investigators, including our laboratory, are focused on the identification of proteins in raw latex and latex products, specifically those responsible for the elicitation of allergic responses. This paper summarizes available information on the mechanism and epidemiology of latex sensitivity and reviews research efforts toward the identification of the antigen(s) responsible for the reactions. The questions of proper diagnosis and testing, heightening awareness, and prevention of reactions are also addressed.

Antitumor activity of phytic acid (inositol hexaphosphate) in murine transplanted and metastatic fibrosarcoma, a pilot study.

We have previously reported that phytic acid (inositol hexaphosphate or InsP6), a natural constituent of cereal diet, when administered in drinking water exerts a consistent antitumor effect on experimental colon cancer in vivo. The objective of this study was to determine whether InsP6 has similar anti-neoplastic effect on other tumor models, such as murine fibrosarcoma. We report that intraperitoneal injection of InsP6 reduces growth of subcutaneously transplanted fibrosarcoma (FSA-1) in mice, prolongs survival of tumor-bearing mice and reduces the number of pulmonary metastases. Since InsP6 is a common constituent of our diet and has very little or no toxic effects, in addition to being chemopreventive, it could have potential use in therapy of cancer as well.

Adjuvant immunotherapy in melanoma: a new approach.

Patients with metastatic cutaneous melanoma to two or more regional lymph nodes have an extremely poor prognosis despite radical lymphadenectomy. In an attempt to improve the survival and to determine the safety of a new method of tumor specific adjuvant immunotherapy in such a high risk group of patients, nine patients were studied. Three to four weeks after regional lymphadenectomy, each of them received a single intradermal injection of Bacillus Calmette-Guérin. Three weeks later, they were immunized by allogenic melanoma cells obtained from live donors with distant metastases. Each patient received three vaccinations, each from a different donor (except in one), to avoid development of HLA response, but maintaining exposure to melanoma antigens. No cultured melanoma cells were used. Each vaccine consisted of mitomycin-C treated tumor cells mixed with purified protein derivative (PPD) of tuberculin given intradermally once per month for 3 months. The patients were then observed with no further treatment. Utilizing the leukocyte migration inhibition test, there was some in vitro evidence of tumor specific cell mediated response which seemed to disappear 1-2 months postimmunization. At 5 years, five of the nine patients (55%) were alive free of disease. No autoimmune diseases were detected in any of the immunized patients. A major hindering factor for such an approach was the limited availability of the allogenic melanoma cells.

An immunological aspect of melanoma and its potential application in adjuvant therapy.

In an attempt to understand some of the natural immunological characteristics of cutaneous melanoma so that we can plan justifiable immunotherapeutic approaches, 23 patients were studied. The autologous leukocyte migration inhibition assay was utilized to assess in vitro their tumor-specific cellular immunity. This assay was specifically used because when presensitized lymphocytes are exposed to the same antigen, they release lymphokines which inhibit the natural migration of the leukocytes. All the patients were staged pathologically according to the TNM system. Twelve of them had regional metastasis, i.e., stage III, and underwent regional lymphadenectomy. The other 11 had distant metastases, i.e., stage IV disease, and all their gross tumors were resected. These 23 patients were the source of tumor material and peripheral blood. Fresh autologous leukocytes were obtained for the assays, from each patient, on the day of surgery and prior to the administration of the preoperative medications. These were tested in vitro, on the same day, with freshly prepared autologous tumor extracts as the source of autologous tumor antigens. The results revealed that the preoperative leukocytes of patients with stage III melanoma expressed hypersensitivity to their tumors, with significant inhibition of their leukocyte migration, compared to those with distant metastases who expressed no such sensitivity, P = 0.012. Such hypersensitized lymphocytes may be capable of producing more efficient lymphokine activated killer (LAK) cells for an effective adjuvant adoptive immunotherapy.

Inositol-phosphate-induced enhancement of natural killer cell activity correlates with tumor suppression.

In recent studies, we have demonstrated that inositol hexaphosphate (InsP6) inhibits experimental colon carcinogenesis. Since natural killer (NK) cells are involved in tumor cell destruction, we investigated the effect of InsP6 on murine NK cell activity. We show that; (i) 1,2-dimethylhydrazine (DMH), a colon carcinogen, depresses NK activity; (ii) in vivo treatment of mice with InsP6 enhances baseline NK activity and reverses DMH-induced depressed NK activity with an inverse correlation (r = -0.9811) with tumor incidence, (iii) short-term in vitro treatment of spleen cells and NK-enriched fraction with InsP6 also enhances NK cytotoxicity in a dose-dependent manner, (iv) inositol potentiates the action of InsP6. Our data suggest yet another important role of inositol phosphates in the regulation of cellular activity.

Anti-tumor activity of recombinant tumor necrosis factor on mouse fibrosarcoma in vivo and in vitro.

The experiments presented were designed first to determine the effects of rTNF on the methylcholanthrene-induced fibrosarcoma (FSA-1) in C3H/JSed mice and second to determine whether the observed effects are the result of direct action by rTNF on the tumor or whether rTNF acts as a mediator of other effector mechanisms. Mice received syngeneic FSA-1 fibrosarcoma cells either s.c. or i.v. in order to evaluate growth of transplantable solid tumor or lung metastases, respectively. The range of dosages, from 10(2) to 2 x 10(5) U of rTNF, was administered i.v. at different intervals after the tumor cell injection. Early injection of 10(3) to 10(4) U of rTNF reduced the growth of s.c. injected tumor and the number of lung metastases in i.v. injected mice. In both cases, survival of mice was also prolonged. However, in vitro treatment of FSA-1 tumor cells with rTNF did not result in the reduction of their proliferating activity after injection into mice, although direct cytostatic and moderate cytotoxic activity of rTNF in vitro was demonstrated. To identify whether other cellular mechanisms are involved in the effects observed in vivo, the anti-tumor activity of rTNF-treated spleen cells was evaluated in vitro using a 75Se release assay. Whereas nontreated spleen cells demonstrated very low cytotoxic activity in this system, the cells from rTNF-treated mice showed marked increase in the cytotoxicity against syngeneic tumor cells. These results suggest that the anti-tumor activity of rTNF represents a combination of its direct effect on tumor cells and indirect effects involving host immune mechanisms.

Thymic factor-induced reduction of pulmonary metastases in mice with FSA-1 fibrosarcoma.

The biological activities of two thymic factors, serum thymic factor thymulin normally present in serum and thymosin alpha-1 (Ta-1) extracted from the thymus gland, have been studied. The effects of the factors on the growth of pulmonary metastases and survival of mice were evaluated in pathogen-free C3H/fSed males. Mice were injected i.v. with the single cell suspension of the syngeneic methylcholanthrene-induced fibrosarcoma. The treatment with thymulin and Ta-1 started two days after injection of 5 x 10(4) to 2 x 10(5) tumor cells per mouse. Different doses of the thymic factors were administered S.C. in sets of 5 daily injections through a period of 2 or 3 weeks. Numbers of tumor colonies in the lung were determined two weeks after the cell injection. Treatment with 0.1 micrograms Ta-1 per injection through the period of two or three weeks, prolonged the survival of tumor-injected mice. Similar effects were observed in mice treated with 0.01 microgram thymulin per injection. Numbers of tumor colonies in lungs of these mice two weeks after the cell injection were also reduced in comparison with saline-treated controls. These findings correlated with prolonged survival time of identically treated mice. The effectiveness of thymic factors in reducing tumor growth was dependent on the tumour load. In addition, the effects induced by Ta-1 persisted longer than observed in thymulin-treated mice. Mice challenged 150 days after the primary tumor cell injection and treatment with Ta-1 demonstrated increased resistance to tumor, while mice treated with other factors behaved as saline-treated controls. The results indicate that both factors exert beneficial effects against tumor growth, although mode of action for each factor may be different.

Effects of radiation therapy on T-lymphocyte subpopulations in patients with head and neck cancer.

Cellular immunity was assessed in 85 patients with head and neck cancer with monoclonal antibodies to lymphocyte surface antigens that identify total T cells, helper cells, and suppressor cells. The control group consisted of 22 healthy volunteers. Nine patients who had surgical procedures for benign diseases were also studied. Compared with the controls, the patients with cancer who received radiation therapy had a significant decrease in total lymphocytes, T cells, helper cells, suppressor cells, and decreased helper/suppressor cell ratio. Significant decreases in lymphocyte subpopulations were not detected in patients tested before treatment or in patients treated with surgery alone. The immune deficits observed were prolonged in duration, with some present in the patients studied up to 11 years after radiation therapy. This long-lasting immune depression may have relevance to tumor recurrences and second primaries in patients with head and neck cancer treated by radiation therapy and to attempts at increasing cure rates with adjuvant agents that improve immune reactivity.

Thymosin alpha 1-induced modulation of cellular responses and functional T-cell subsets in mice with experimental autoimmune thyroiditis.

The effects of Ta-1, a peptide constituent of thymosin fraction 5, were studied on murine autoimmune thyroiditis using two congenic strains of mice, B10.Br (Br) and B10.D2 (D2), which are sensitive and resistant to experimental autoimmune thyroiditis (EAT) induction, respectively. EAT was induced by either 2 weekly iv injections of mouse thyroglobulin with adjuvant lipopolysaccharide (LPS) or intradermal injection of thyroglobulin mixed with complete Freund's adjuvant (CFA). The criteria for induction and intensity of thyroiditis were the level of lymphoid infiltration in the thyroid gland and the titer of anti-thyroglobulin antibodies. Ta-1 was given in 5 or 10 daily sc injections in doses ranging from 0.0001 to 0.1 microgram/injection. The injections were commenced at varying intervals from the 1st to the 4th week after immunization. T-Cell subsets in the spleens were determined 2 weeks after the first antigen injection and thyroid infiltration was determined 3 weeks later. Treatment with Ta-1 between the two antigen injections increased the level of thyroiditis in resistant mice, but had no effect in sensitive mice. Treatment for the first 2 weeks had similar effects in resistant mice, but also suppressed thyroiditis in the sensitive strain. Later treatments, during the 3rd and 4th weeks after immunization also revealed immunomodulating properties of Ta-1, with a suppressing effect on thyroiditis in sensitive mice and an enhancing effect in the resistant strain. Both effects of Ta-1 were dose dependent. The effects of Ta-1 on the individual phenotypes were also dose dependent. The dose of 0.01 microgram greatly lowered the percentages of Lyt-2+3+ cells in D2 mice and mildly increased the percentages in Br mice, but did not change the Lyt-1+ cell level in either strain. On the other hand, the dose of 0.001 microgram greatly increased the percentage of Lyt-1+ cells in D2 mice and mildly decreased it in the Br strain, but did not alter the Lyt-2+3+ cell subset in either strain. Thus, both doses of Ta-1 modulated Lyt-1+/2+3+ ratios, with each dose affecting a different T-cell subset. The changes in the response to thyroglobulin are apparently exerted through the regulation of the functional T-cell subset balance.

Experimental autoimmune thyroiditis: modulation of the disease level in high and low responder mice by thymosin.

Experimental autoimmune thyroiditis (EAT) in mice is characterized by production of anti-thyroglobulin autoantibodies and lymphoid infiltration of the thyroid gland. The pathogenesis of EAT is genetically controlled, but its mechanism is not yet clear. To investigate the mode of the expression of genetic control in the development of the disease, we attempted to modulate immune responses with thymosin fraction 5 (T-5) in mice which are either high or low responders to thyroglobulin. Severe thyroid lesions in high responder mice appear 2-3 weeks after immunization, while low responders develop very mild or no lesions. High responder mice have also higher antibody titer than the low responder strain. T-5 administered in five daily injections before or simultaneously with immunization, was strongly suppressive to EAT development in the high responder strain while there was no effect on thyroiditis level in low responder mice. T-5 also decreased the severity of thyroiditis in high responder mice when injected 2-4 weeks after immunization. In low responder mice, however, the same treatment increased the thyroiditis level. The effects of late injection of T-5 were dose-dependent. In all experiments, the antibody titres were not affected by T-5 treatment. The results demonstrate the ability of T-5 to modulate development and intensity of EAT. The contrasting effects of T-5 in high and low responder mice illustrate the relationship between the effects of T-5 and host immune status prior to study, suggesting that the level of immunoregulatory cell activity may differ in these two strains.

Adriamycin induced immunomodulation: dependence upon time of administration.

The immunomodulating capabilities of the anti-neoplastic agent, Adriamycin, were investigated. The day of Adriamycin administration to mice was varied from -15 to -1, day 0 being when mice were either immunized or sacrificed and their spleen cells sensitized in culture. Humoral and cellular immune responses against allogeneic or xenogeneic cellular antigens in mice and in culture, as well as phagocytic and ADCC activities were evaluated using spleen cell populations. The cellular responses and phagocytic activities were affected in a cyclical manner with time after Adriamycin's administration. Peaks of increased activity were seen subsequent to day -5 and day -11, administration and low activities following day -1, -3 and day -7, -9 administration. The humoral responses were not affected in a biphasic manner but single peaks of increased activity were seen which corresponded to the times of low cellular cytolytic and phagocytic activities. The ADCC was independent of time of Adriamycin administration. The significance of these findings to the design of therapeutic protocols is discussed.

Augmentation of the development of immune responses of mice against allogeneic tumor cells after adriamycin treatment.

In C57BL/6J mice, depending on the dose of P815 cells used for immunization, Adriamycin exerted different effects on the cell-mediated lytic response and complement-dependent cytotoxicity. At the dose of 3 X 10(7) P815 cells, Adriamycin treatment had no apparent effect on cell-mediated lytic response regardless of timing of drug treatment. At lower doses of antigen (10(7) or 5 X 10(6) cells), the response was augmented in Adriamycin-pretreated mice. Similarly, under conditions which led to a suboptimal complement-dependent humoral response of untreated control, Adriamycin pretreatment resulted in an augmented response; under conditions of maximal response, Adriamycin was suppressive. Suppression was maximal if the drug was injected at either the same time or shortly before or after antigen. The cell-mediated lytic response was proportional to the dose of antigen used, while the complement-dependent humoral lytic response was inversely proportional to dose of antigen in the range used in these experiments. Secondary cell-mediated lytic response in culture was also augmented if mice had been pretreated with Adriamycin 5 days before the primary immunization. The cell-mediated lytic response of spleen and peritoneal exudate cells from mice immunized with relatively low doses of P815 cells 5 days after treatment with Adriamycin was increased 12 to 15 days after immunization. The cytotoxic effects were present in both plastic adherent and nonadherent fractions of either spleen or peritoneal cell populations. All these effector cells were found to be anti-Thy 1.2 sensitive. The phagocytic activity of spleen cells was increased after immunization, but no drug effect was observed; following 24 hr of culture, however, cells from drug-treated immunized donors had increased phagocytic activity as compared to that of controls. Increased phagocytosis also developed in cells nonadherent to plastic.

Selective imbalances of cellular immune responses by adriamycin.

It has been demonstrated that spleen cells from mice treated with adriamycin not only develop an increased cell-mediated immune response during culture with allogeneic tumor cells, but also have increased phagocytic activity following culture, respond to heat-treated (45 degrees C) alloantigen, develop an increased suppressor cell function, and are less sensitive to a suppressor cell activity. Thus, changes in spleen cell subpopulations occurring in the donor mice consequent to drug treatment result in demonstrable selective imbalances of cellular functions involved in the immune response. One cell type which has been implicated as being necessary in the expression of all these functions is the monocyte-macrophage, and it is suggested that an effect of adriamycin on progenitors of this cell type may lead to the imbalances.

Autoimmune murine thyroiditis IX. Relationship of humoral and cellular immunity to thyroiditis in high and low responder mice.

Good responder (C57/Br/cd) and poor responder (C57BL/10) mice were immunized with mouse thyroid extract in Freund's complete adjuvant. The good responder mice first showed antibody to thyroglobulin on the seventh day while the poor responder animals had slightly lower titers and antibody did not appear until day 12. The first signs of thyroid infiltration appeared on day 4 in the responder strain, and severe lesions were seen between the third and fifth week. In contrast, the poor responder mice showed almost no evidence of infiltration. The macrophage disappearance reaction, a measure of cell-mediated immunity, was similar in the responder and nonresponder strains.

The macrophage disappearance reaction (mdr) as an in vivo test of delayed hypersensitivity in mice.

The macrophage disappearance reaction (MDR), as an invivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 h after immunized mice received an intraperitoneal injection of 200 microng of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.