ImuVert, a sterile preparation composed primarily of Serratia marcescens membrane vesicles and ribosomes, was significantly inhibitory to murine cytomegalovirus (MCMV) infections in BALB/c mice. Antiviral activity was manifested as increased survivor number and decreased recoverable virus titers in spleens, lungs and salivary glands. Treatments were intraperitoneal (i.p.) beginning 24 h pre, 4 h post- or 24 h post-virus inoculation and then repeated 4 days later. Doses of 5, 16 or 50 micrograms/mouse were effective; 160 micrograms/mouse, which caused host weight loss in toxicity controls, was not inhibitory to the infection. A single i.p. treatment of mice substantially augmented natural killer (NK) cell activity and increased total B-cells, while reducing total T- and T-helper cells. A late (48 h) decline in T-cell function and transient increases in B-cell function were observed in the treated animals. Serum interferon was not induced. Mice pretreated with anti-asialo GM1 antibody to reduce their NK cell populations, then infected with MCMV and treated with ImuVert were protected to the same degree as normal animals. Severe combined immunodeficient mice infected with MCMV and treated with ImuVert were not protected from the infection. These data suggest ImuVert to act by a mechanism other than NK cell activation in preventing MCMV infections.

Tumor-specific cytotoxicity was measured in fresh human biopsy specimens by a modification of the differential staining cytotoxicity assay. ImuVert, a cytokine inducer derived from Serratia marcescens, which produces broad-spectrum activation of both macrophages and lymphocytes, was dramatically more effective when it was tested in tumors obtained from patients with previously treated, chemotherapy-responsive adenocarcinomas (breast and ovary) than when it was tested in tumors obtained from either previously untreated patients or previously treated patients with chemotherapy-refractory adenocarcinomas (colon, lung, pancreas, stomach, kidney, gallbladder, uterus, and prostate). Similar findings, relating to prior chemotherapy treatment status, were obtained for tumor necrosis factor and interferon gamma, but not for interleukin-2 or interferon alpha. On the basis of these findings and on other evidence in the literature, we speculate that response to chemotherapy produces massive release and processing of tumor antigens. We further speculate that this response leads to a state in which the human immune system is primed (via in situ vaccination) to respond to exogenous macrophage-activation signals with potent, specific antitumor effects.

Endotoxins represent a family of ubiquitous bacterial lipopolysaccharides found in water and raw materials. These substances have the ability to generate interleukin-1 (IL-1) and induce fever, as well as other acute phase phenomena. A study was undertaken to determine levels of background endotoxin in (1) continuous ambulatory peritoneal dialysis solution, (2) spent dialysate subsequent to overnight dwell, (3) hemodialysis solution, and (4) Limulus amebocyte lysate-reactive material (LAL-RM) in hemodialyzers and patient plasma. Levels of endotoxin in all of the above cases were less than thought to be required to induce biological activity, such as pyrogenicity, through IL-1 generation. Although nanogram amounts of LAL-RM are associated with some hollow-fiber membranes as well as the plasma of patients on those membranes, this material per se does not appear to produce IL-1 in vitro.

Sera from 138 patients who had experienced hypersensitivity-type reactions while on hemodialysis (reactors) were examined retrospectively by the radioallergosorbent test (RAST) for specific IgE antibody to ethylene oxide (ETO). Seventy-eight hemodialysis patients without a history of reaction were also evaluated as controls. Elevated serum RAST values (greater than 2.0) were more common in reactors (63%) than in controls (11%, p less than 0.001). In a second study, RAST assays were performed using human serum albumin conjugated to ETO (HSA-ETO) as antigen and also using a concentrate of fluid used to rinse ETO-sterilized dialyzers ("dialyzer extract") as antigen. The RAST ratios obtained with HSA-ETO were similar to those obtained using the dialyzer extract (rank order correlation coefficient = 0.829, p less than 0.001). In a third study, RAST inhibition was demonstrated both by HSA-ETO and dialyzer extract. Our results, extending previously published reports, suggest that hypersensitivity to ETO might play an important role in hemodialysis-associated hypersensitivity-type reactions.

Hypersensitivity-like reactions are an infrequent but persistent clinical manifestation of hemodialysis. Although a Limulus-amebocyte-lysate-reactive material (LAL-RM) has been demonstrated in some hemodialyzers, its potential role in causing anaphylactoid reactions has not been addressed heretofore. In this chapter, current knowledge about LAL-RM will be presented and integrated with the major mechanisms generally recognized to induce hypersensitivity-type reactions. These mechanisms include: classical induction of allergy by IgE and classical complement activation by IgG and IgM. Alternative complement activation will be not discussed as it has been extensively reported in the literature of hemodialysis and because LAL-RM has not been shown to activate C3a extensively.

Repeat plasma donors were studied to determine whether there was a relationship between allergic‐type reactions during plasmapheresis and IgE‐dependent sensitization to ethylene oxide gas used for sterilization of disposable fluid administration sets. Serums from 32 donors with allergic‐type reactions and 84 donors who had no reactions but were exposed to the same materials and served as controls were tested for IgE‐antibodies to ethylene oxide. The results, expressed as an IgE ethylene oxide index, were greater than 2 in 78 percent of serums from donors with allergic reactions and 12 percent of serums from controls. This association was significant (p < 0.0001). Reactivity of the antibodies was directed against an ethylene oxide‐ human serum albumin conjugate and not against human serum albumin carrier protein. IgG antibodies with ethylene oxide specificity also were present in the serums of repeat plasmapheresis donors. Each of seven rabbits immunized with an ethylene oxide‐protein conjugate responded with a high serum level of antibody with ethylene oxide specificity. It was concluded that the residual ethylene oxide in fluid administration sets is immunogenic and may cause allergic reactions in plasma donors.

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.

We observed immediate-type hypersensitivity reactions in 6 of 600 donors who underwent automated plateletpheresis procedures. Ethylene oxide gas, which was used to sterilize plastic components in the disposable apheresis kits, represented a possible source of sensitization. In skin-prick testing, 4 of the 6 donors who had had a hypersensitivity reaction and none of 40 controls who had not had such a reaction had positive tests when an ethylene oxide-human serum albumin reagent was used (P less than 0.001). Radioallergosorbent testing revealed that serum from 4 of the 6 donors who had reactions, but from only 1 of 145 controls, contained IgE antibodies to ethylene oxide-albumin (P less than 0.001). All six of the donors who had reactions had specific ethylene oxide-induced basophil histamine release (mean release [+/- SE], 34.2 +/- 5.6 percent), whereas none of four controls had specific histamine release in response to ethylene oxide (mean release, 5.7 +/- 1.2 percent; P less than 0.005). Repeat plateletpheresis in five of the donors who reacted was accompanied by a recurrence of mild allergic symptoms. These studies demonstrate that sensitization to ethylene oxide gas can occur in healthy plateletpheresis donors and that it may result in immediate hypersensitivity reactions during the donation. The prevalence of such reactions was 1.0 percent in our apheresis donor population.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.

Recent clinical evidence suggests that papain and chymopapain may share common allergenicity. Patients that become sensitized to papain may subsequently experience an allergic reaction when they are exposed to chymopapain. This study demonstrates a cross antigenicity between the proteolytic enzyme preparations papain and chymopapain. Serum samples from six patients who demonstrated 4+ skin reactions to papain also revealed positive RAST ratios to both papain and chymopapain. In addition, serum samples from 12 clinically nonreactive patients who had discolysis with chymopapain demonstrated positive RAST results to papain as well as to chymopapain.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.

In patients receiving long-term hemodialysis (HD), we have examined the presence of IgE-dependent sensitization to ethylene oxide (EO) gas, which is used for sterilization of disposable medical products including dialyzers. Serum was obtained from 25 patients who experienced acute allergic reactions during HD, five patients receiving HD with isolated eosinophilia, and 37 unselected patients receiving HD. Sera from 22 of 25 of the allergic reaction group and from five of 35 of the unselected group were demonstrated to contain IgE antibodies with specificity for EO. Corresponding IgG antibodies were also present. No such antibodies were detected in serum from normal controls or ragweed-allergic patients. The serum from one patient with isolated eosinophilia had a borderline elevated IgE antibody level. These results demonstrate a close relationship between the presence of IgE antibodies to EO and HD-related allergic reactions in this patient population.

Discolysis (chemonucleolysis) with chymopapain, the proteolytic enzyme, is currently being used in the treatment of herniated lumbar discs. Of the patients receiving treatment, approximately 1% experience an anaphylactic reaction. This reaction is thought to be mediated by IgE antibodies to chymopapain. Because of this, it may be possible to predict potential reactors using in vitro testing. In the current study, five out of seven patients who experienced allergic anaphylaxis during discolysis demonstrated high IgE serum levels to chymopapain before treatment.