Equal allergenic potency of beta-lactam antibiotics produced by chemical or enzymatic manufacturing--mouse IgE test.

BACKGROUND: Cephalexin and amoxicillin are semisynthetic beta-lactam antibiotics with a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms. Both antibiotics are produced by a new 'green' process in which enzyme technology is used to combine the intermediate structure and the side chain in an aqueous medium to yield cephalexin or amoxicillin, thus avoiding the use of several chemical reagents and volatile organic solvents. As a result of the enzyme technology a new residual protein impurity has been identified. To check for the sensitizing capacity of the residual protein, a mouse IgE test was used to detect differences in the production of specific IgE by chemical or enzymatic preparations of the antibiotics. METHODS: Balb/c female mice were immunized intraperitoneally with alum and conjugates of different amoxicillins or cephalexins with ovalbumin (OVA). After 16 days, the amoxicillin mice were injected with one half the original amount of antigen. After 19-23 days, the sera were tested for specific IgE by the passive cutaneous anaphylaxis assay in Sprague-Dawley rats. The greatest dilution of sera which resulted in a positive response was the titer of specific IgE. RESULTS: No significant differences were found between the titers of specific IgE caused by the chemically and enzymatically produced beta-lactam antibiotics, indicating that the antibiotics are equal in allergenicity. CONCLUSIONS: The data show that a residual level of 35 ppm protein did not affect the allergenic potency of these beta-lactam antibiotics as determined by the mouse allergenicity model.

Immunomodulatory screening test of corn oil administered orally to female mice: effect of timing of dosing within 24 hours.

The effect of varying the dose-delivery time within a 24 h period (12:12 light-dark cycle) on the immunomodulatory properties of corn oil administered by gavage to 120 B6C3F1 female mice was investigated. Mice, housed in six separate boxes equipped with timers to regulate light onset and offset (staggered by 4 h increments), were treated for 5 consecutive days by intragastric (i.g.), administration of 5 mL/kg corn oil. Negative and positive control mice were given sham injections (needle inserted, but no injection). Sheep red blood cells (SRBCs) were injected intraperitoneally (i.p.) on the fifth day. Three days later, positive control mice were given cyclophosphamide intraperitoneally (80 mg/kg). Four days after SRBC injection, mice were weighed and killed, and spleens and thymuses were removed and weighed. Spleens were brought to single-cell suspensions and tested for an antibody response to the SRBC. Plaque-forming cells (PFCs), as measured per spleen, per 10(6) viable spleen cells or per 10(6) total spleen cells, exhibited significant circadian rhythms for mice given corn oil, but not for sham-gavage- and cyclophosphamide-treated mice. The peak response (acrophase, phi) occurred at 21 h, 22 h, and 23 h after lights on (HALO), respectively, with PFC values significantly different between the different time points. Corn oil and sham gavage affected the circadian pattern of antibody production; there was a high-amplitude (21-27%) rhythm observed when mice were treated with corn oil and no rhythm when mice received the sham-gavage treatment. In addition to testing mice near the end of the daily dark span and/or early light span to obtain a maximum immune response, this finding points to the importance of including as controls a group of animals that are not treated at all and a group given vehicle alone, rather than only sham-treated animals, for comparison with experimentally treated animals.

The influence of vehicle gavage on seasonality of immune system parameters in the B6C3F1 mouse.

Seasonal hyporesponsiveness and other immune system variations were observed in female B6C3F1 mice during routine screening tests for immunomodulation. In a retrospective assessment, 4 years of data from over 1200 naive, vehicle, and immunosuppressed (cyclophosphamide-treated) control mice were compiled and analyzed for uniformity and significant circannual pattern of immune response. Endpoints included body, spleen, and thymus weights and an immunotoxicity assessment which enumerates specific antibody plaque-forming cells (PFC) in the spleen following immunization with sheep red blood cells. Dosing vehicles were water, corn oil, or 1% methyl cellulose instilled by oral gavage in a 5-20 ml/kg volume once daily for 5 days. Four days later, terminal organ and body weights were recorded and PFC were quantitated. Upon analysis, individual datapoints were arrayed in consistent circannual and seasonal patterns. In naive mice, the yearly peak response in circannual rhythm (acrophase) for body weight and PFC parameters occurred in the summer, with acrophases for spleen and thymus weights located in the spring. Vehicle gavage modulated the circannual/seasonal means and acrophases of all measured endpoints in distinct patterns which varied by vehicle. Body weight was the endpoint least affected by vehicle treatment. Corn oil was the vehicle resulting in the most dramatic effects on natural rhythm. As expected, the naive mice receiving an ip injection of cyclophosphamide exhibited significant decreases (p

Successful treatment of herpetic infections by autohemotherapy.

Herpes zoster (shingles) affects a significant number of individuals over age 50. To date, no satisfactory treatment has been available. The clinician author (JHO) witnessed a dramatic response of a shingles patient to autohemotherapy: the pain was completely relieved and lesions gone within 5 days with no recurrence of either. Treatment of other herpetic patients then began with autohemotherapy. Twenty-five patients with herpes were given an autologous blood transfer of 10 mL of blood from the antecubital vein into the gluteal bundle and followed for clinical signs. A 100% favorable response occurred in 20 patients who received autohemotherapy within 7 weeks of the onset of clinical signs and 1 other who received autohemotherapy at a 9-week interval. No untoward signs or symptoms of the treatment occurred. Autohemotherapy has been demonstrated to be effective in elimination of clinical sequelae in these cases of herpes infections and these results justify further rigorous clinical investigation.

Immune function and host defense in rodents exposed to 60-Hz magnetic fields.

This study was conducted to evaluate the influence of subchronic exposure to pure, linearly polarized 60-Hz magnetic fields (MF) on the host immune response in mice. The experimental design was as follows: three groups were exposed continuously (18.5 hr/day) to MF at field strengths of 0.02, 2, or 10 gauss (G), one group was exposed intermittently (1 hr on/1 hr off) to MF at a field strength of 10 G, and one group served as a sham control. Experimental endpoints included spleen and thymus weights and cellularity, antibody-forming cell (AFC) response, delayed-type hypersensitivity (DTH) response, splenic lymphocyte subset analysis, susceptibility to infection with Listeria monocytogenes, and natural killer (NK) cell activity. No differences in body weight, lymphoid organ weight, or lymphoid organ cellularity were observed in any MF-exposed group in comparison to sham controls. Likewise, no statistically significant differences were found in comparisons of AFC responses. Isolated statistically significant differences from control were observed in MF-exposed mice in the DTH assay, although no clear dose-related pattern of altered activity was seen. Splenic lymphocyte subset parameters examined were within normal limits in all groups, and no differences between control and MF-exposed mice were found. Host resistance to bacterial infection was not altered at any MF exposure examined in this study. Finally, although apparently dose-related, statistically significant alterations were observed in an initial study of NK cell function, repeat studies failed to demonstrate a consistent pattern of alteration.

Local versus systemic immunotoxicity of isobutyl nitrite following subchronic inhalation exposure of female B6C3F1 mice.

Female B6C3F1 mice were exposed to isobutyl nitrite (IBN) by inhalation at 0, 37.5, 75, or 150 ppm for 6 hr per day, 5 days per week for 15 weeks. The potential of this compound to induce immunotoxicity was assessed during the 3rd, 13th, 14th, and 15th week of exposure and after 2 weeks of recovery following the 15 weeks of exposure. Both systemic and lung immune functions were examined, including body and lymphoid organs weights, pulmonary macrophage function and host defense, expression of splenic lymphocyte cell-surface markers, natural killer cell function, mixed lymphocyte reaction, and induction of specific antibody to a T-cell-dependent antigen. There was a dose-related suppression of T-cell-dependent antibody-forming cell responses in the spleen following IBN exposure; however, other measures of T-cell and nonspecific immunity were not significantly affected. A dose-related increase of H202 production by alveolar macrophages was present after 12 but not after 68 exposures to IBN. In contrast, pulmonary host defense mechanisms against Klebsiella pneumoniae were unaffected. These results suggest that in the absence of changes in host resistance, IBN may have selective and partially reversible effects on the immune system.

Immunotoxicologic effects of ethylene dibromide in the mouse and their modulation by the estrous cycle.

Estrous cycle modulation of immunologic sensitivity to ethylene dibromide (EDB) was studied in addition to toxicologic end points. Female B6C3F1 mice were injected intragastrically with 31.25, 62.5, or 125 mg/kg EDB for 5 days a week for 12 weeks. Vaginal smears determined the estrous cycle. At 125 mg/kg there were decreases in hemoglobin and hematocrit and longer estrous cycles (5.5 vs 4.3 days, p = 0.006), and increases in cholesterol, triglycerides, total protein, and albumin. The negative dose response seen for T- and B-cell mitogenesis around metestrus was absent for mice near estrus. The high dose of EDB prolonged intervals between estrus, was immunotoxic and immunosuppressive.

Optimization and validation of an ELISA to measure specific guinea pig IgG1 antibody as an alternative to the in vivo passive cutaneous anaphylaxis assay.

Assessment of the allergenic potency of enzymes involves the use of a guinea pig model in which specific IgG1 antibody titers are used as the endpoint. The in vivo passive cutaneous anaphylaxis (PCA) assay is used to measure specific IgG1 antibody. This report describes the development and validation of an enzyme-linked immunosorbent assay (ELISA) to measure guinea pig specific IgG1 antibody as an in vitro alternative to the PCA assay. Cross reactivity of various rabbit and mouse (monoclonal) anti-guinea pig IgG1 preparations were evaluated using purified IgG1 and IgG2 from serum of guinea pigs immunized with ovalbumin. The two subclasses of guinea pig IgG were purified by first using Protein A affinity chromatography, followed by anion exchange chromatography and fluid phase isoelectric focusing. Affinity-purified rabbit anti-guinea pig IgG1 was shown to have minimal cross reactivity toward IgG2, while providing a strong signal with IgG1. The ELISA was designed as an antigen capture system in which the following are added in sequence: (1) enzyme antigen (passively adsorbed to the plate), (2) diluted serum samples from guinea pigs immunized with enzyme, (3) affinity-purified rabbit anti-guinea pig IgG1, (4) alkaline phosphatase-conjugated donkey anti-rabbit IgG, and (5) p-nitrophenyl phosphate substrate. Three replicate ELISA and PCA analyses were conducted on sera samples of varying titers from guinea pigs immunized with either Alcalase (protease), BPN' (protease), and Termamyl (amylase) enzyme. The correlation coefficients (r2) between the ELISA and PCA assay for Alcalase, BPN', and Termamyl were 0.826, 0.945, and 0.755, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethylene dibromide: evidence of systemic and immunologic toxicity without impairment of in vivo host defenses.

Ethylene dibromide was administered intragastrically on 14 consecutive days to B6C3F1 female mice. Host resistance was not altered after challenge with B16F10 tumor cells, Listeria monocytogenes, influenza, or Herpes simplex viruses. In contrast, decreases were seen in relative thymus and spleen weights, red blood cells, hemoglobin, hematocrit, and in alveolar macrophage, natural killer cell, T-cell, and mixed lymphocyte culture responses. Increases occurred in relative kidney and liver weights, cholesterol, peripheral neutrophils, resident peritoneal exudate cells (with increased phagocytosis) and plaque-forming cells. There was little difference between the dose that caused immune modulation and that which produced significant toxicity.

An immunotoxicity assessment of food flavouring ingredients.

A rapid screening protocol incorporating key elements of the US National Toxicology Program's immunotoxicity tier testing strategy was used to evaluate the effects of 35 commonly used food flavouring ingredients on humoral and cell-mediated immune responses. The test compounds were administered intragastrically on a daily basis for 5 days at three dose levels to female CD-1 or B6C3F1 mice, 6-8 wk old. A host resistance assay (Listeria monocytogenes bacterial challenge) was conducted to assess cell-mediated immunity. Humoral immunity was measured by the antibody plaque-forming cell (PFC) response to sheep erythrocytes. Body weights, lymphoid organ weights and spleen cellularity were also measured. Cyclophosphamide (80 mg/kg) served as an immunosuppressive positive control agent. The results indicated that the majority of the flavouring ingredients tested did not modulate the cell-mediated or humoral immune response. However, at very high dose levels, two of the materials tested, peppermint oil and citral dimethyl acetal, did increase mortality rate and reduce survival time in the host resistance assay. Neither of these materials significantly altered the PFC response. This rapid, economical screening battery for potential immunotoxicants proved to be a useful means of evaluating a large number of structurally diverse compounds and mixtures to prioritize them for more definitive testing.

Evidence for genetic basis of seasonal differences in antibody formation between two mouse strains.

In order to confirm the presence of an acrophase difference based upon genotype in the seasonal expression of an immune competence end point, splenic plaque-forming cell (PFC) response to sheep red blood cells (SRBC), female B6C3F1 and CD1 mice were concurrently studied for PFC response during two studies performed in each season for 1 year. Mice were multiply housed, fed ad libitum, and standardized to light (06:00-18:00); dark (18:00-06:00). For each strain and study, subgroups were either naive (n = 10), received a vehicle (n = 10) or Cytoxan (n = 5). Challenge with SRBC occurred in early afternoon 4 days before harvesting of spleens and PFC assay. All other procedures were performed early in the daily light span. Analysis of variance and single cosinor analysis revealed a significant seasonal time effect for PFC in naive mice of both strains. Antibody formation was greatest in spring for CD1 mice and in summer for the B6C3F1 mice. These acrophases were consistent with earlier results for both strains and show the phenomena to be reproducible and genetically based.

Surgical influence on murine immunity and tumor growth: relationship of body temperature and hormones with splenocytes.

Adrenalectomy predisposed the C3HeB/FeJ Mouse to tumor from a low dose of tumor cells, derived from a C3H spontaneous mammary adenocarcinoma. Sham surgery had a similar effect. In contrast, ovariectomized females, intact females, and male mice did not allow the low dose of cells to develop into a tumor. In order to better understand the role of hormones on the immune system controlling tumor growth, normal C3HeB/FeJ mice were studied for the effect of corticosterone or estradiol on splenic lymphocyte surface antigen expression. Adrenalectomy and ovariectomy caused a decrease in the percentage of all T cell subclasses and an increase in absolute numbers of immunoglobulin-bearing cells. Reconstitution of ovariectomized mice with estradiol did not significantly alter lymphocyte cell surface antigen expression. In contrast, injection of corticosterone into adrenalectomized mice brought these values to normal. Further study on normal mice placed on a 12:12-hr light:dark schedule showed that the hours after lights on (HALO) had a significant effect (analysis of variance) on body temperature, percentage of splenic B cells, T pan, T helper and T suppressor cells, and absolute numbers of T pan cells. Brain dehydroepiandrosterone sulfate correlated positively with T pan lymphocytes, but showed no significant effect on HALO. In contrast, body temperature showed a strong circadian rhythm (P less than 0.001). In addition, the presentation of estrus was circadian rhythmic (P = 0.003) with 58% of mice in estrus at 16 HALO and only 8% at 4 HALO. Multiple regression analysis revealed body temperature was strongly associated with absolute numbers of splenic T lymphocytes and their subsets, as well as percentage of B lymphocytes, Thy 1.2-, and Lyt-2-bearing cells. Similarly, HALO and estrous cycle stage were associated with percentage of T helper cells. The data showed that body temperature and hormones were associated with the cell surface antigens on lymphocytes and suggest that they affect lymphocyte function.

Reproducibility of seasonal effects in antibody formation and host resistance in the outbred mouse.

The purpose of this study was to assess the reproducibility of any seasonal effects in the outbred CD1 mouse of antibody production to sheep red blood cells (SRBCs) and host resistance to the bacterium Listeria monocytogenes. A marked seasonal effect on antibody production was seen when 5- to 6-week-old female CD1 mice were studied on a weekly basis for a period of 2 years. Maintained on a 12:12 h light:dark schedule, animals were held for 12 days prior to experiment to insure physical condition and acclimatization to the lighting regimen. Beginning at 4 h after lights on (HALO) for day 1 and 2 HALO thereafter, groups of mice were (a) not treated, (b) administered a vehicle (corn oil, 1% methylcellulose, or distilled water) by oral gavage for 5 days, or (c) not treated, but given an intraperitoneal injection of cyclophosphamide 24 h prior to assay. On the fifth day, mice were injected with SRBCs intravenously. Four days later, antibody formation against SRBCs was measured using spleen cells. Circannual and seasonal rhythms were displayed by each group of animals, with greatest antibody production, indicated by the number of plaque-forming cells (PFCs)/million viable cells, in the Spring (range of double amplitude = 36-108%). The timing of these rhythms was reproducible from one year to the next. In contrast, the magnitude of the response in year 1 was significantly different from year 2 for animals given vehicle or not treated. Cyclophosphamide-treated mice had consistently low numbers of plaque-forming cells. Host resistance was studied in separate mice treated with vehicles at the same time as the antibody assay. On the third day of dosing, mice were injected intravenously with Listeria monocytogenes (LM) and monitored for death for 10 days. When analyzed by Kruskal-Wallis life table analysis, there was no overall effect of vehicle on survival for 1987 but there was an effect for 1988 and when data from both years were combined. Distilled water-treated mice had lower survival rates than the other two vehicles. Mice treated with distilled water displayed a circannual rhythm for survival for 1988 and for both years combined, in contrast to the other two vehicles. When each vehicle was analyzed separately for seasonal effect, a significant effect of season occurred for corn oil- and distilled water-treated animals. The greatest survival rate and longest survival time generally occurred in the months between July and December.

Effects of inhaled hexachlorobenzene aerosols on rat pulmonary host defenses.

Pulmonary bactericidal activity, macrophage phagocytic activity, alveolar macrophage (AM) enzyme activity, and T- and B-cell mitogenesis of lymphocytes from lung associated lymph nodes (LALN) or mesenteric lymph nodes (MESLN) were assessed in Sprague-Dawley rats exposed 4 hr/d, 4 days/wk for 1, 4, or 16 days to hexachlorobenzene (HCB) aerosols. Pulmonary bactericidal activity was depressed after 1 or 4 but not 16 exposures to 35 mg/m3 of HCB. AM phagocytosis of 51Cr-RBC in vitro was increased after 4 but not 1 or 16 exposures to HCB, and no effect was observed in peritoneal macrophages. HCB significantly enhanced mitogenesis in MESLN to the B-cell mitogen Salmonella typhimurium lipopolysaccharide (STM) after 4 exposures; LALN STM mitogenesis and LALN and MESLN mitogenesis to phytohemagglutinin (PHA) were not affected. After 16 exposures, however, the PHA responses in LALN and MESLN were significantly increased and decreased, respectively.

Assessment of carbamate pesticide immunotoxicity.

Adult female Swiss Webster and B6C3F1 mice received distilled water only or water containing 0.1, 1.0, 10, 100 or 1000 ppb of aldicarb daily for 34 days. The target concentration of aldicarb present in the 10 ppb dosing solution was analytically verified on a daily basis as was its stability over a 48-hr period. To develop an immune profile of this compound, functional parameters measured after exposure included resistance to infectious viral challenge; quantitation of splenic antibody-forming cells to sheep erythrocytes; splenic lymphocyte blastogenesis to B-cell mitogens; and T-cell mixed lymphocyte culture response. In addition, gross and histopathologic examinations of tissues relevant to the immune system were performed. The absence of significant effects on any of these parameters suggests that aldicarb at environmentally relevant exposure concentrations is not immunotoxic in rodents.

Estrous influence on surgical cure of a mouse breast cancer.

We have studied the effect of estrous stage, as reflected by vaginal cellularity, at the time of surgical resection of an estrogen receptor-bearing mammary adenocarcinoma upon the metastatic potential of that tumor in the C3HeB/FeJ mouse. Presence of the tumor prolonged the length of the estrous cycle by approximately 25% and removal of the tumor returned the cycle to its usual duration. Neither estrous stage at tumor implant nor size of tumor at resection (within a small range) had significant independent effects upon differences observed in the incidence of subsequent pulmonary metastases. However, estrous stage at time of surgical removal of the tumor, as reflected by cell types in vaginal smear, markedly affected whether or not metastases ultimately appeared. Because the estrous cycle in mice, comparable to the human menstrual cycle, reflects high-amplitude, rhythmic changes in hormone concentrations, it may be that the hormonal status of a women at the time of tumor resection is an important determinant of whether or not that breast cancer ultimately metastasizes.

Evidence of immunologic control of lung injury induced by trimellitic anhydride.

Trimellitic anhydride (TMA) is a chemical intermediate that has been shown to cause immunologically mediated respiratory syndromes in humans. We developed a rat model in which lung lesions accompanied by TMA-specific antibody resembled effects seen in humans. Two sets of experiments were undertaken to determine if TMA lung injury was primarily controlled by the immune system. Experiment 1: Rats were exposed to 95 micrograms/m3 of TMA 6 h/day, 5 days/wk for 2 wk during which time they received daily injections of either the immunosuppressant cyclophosphamide or saline. The TMA-exposed/saline control rats exhibited the usual TMA-induced lung lesions accompanied by TMA-specific antibody. However, the TMA-exposed/cyclophosphamide rats showed no lesions and no antibody. The spleen cells from all rats were subjected to lymphocyte blastogenesis assays using T- and B-cell mitogens. Results confirmed that cyclophosphamide-treated rats showed very little if any blastogenic response, whereas saline-treated rats gave the normal immune response. Thus, cyclophosphamide eliminated T- and B-cell function, which in turn prevented the occurrence of TMA lesions. Experiment 2: An initial passive transfer experiment showed that serum from TMA-sensitized rats could be adoptively transferred into naive recipient rats, which when given a single TMA inhalation challenge exhibited TMA-induced lesions. Similar attempts to transfer spleen cells or spleen cells plus serum did not predispose recipients for lesions. A second modified passive transfer of sensitized serum using a larger number of recipient rats, followed by a TMA challenge, resulted in lesions in 14 of the 16 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The pathologic and immunologic effects of inhaled acrolein in rats.

Four groups of 40 male Sprague-Dawley rats each were exposed by inhalation to target concentrations of 0, 0.1, 1.0, and 3.0 ppm of acrolein 6 h/day, 5 days/week for 3 weeks. Subsequent changes in local pulmonary immunity were determined by examining the number of antibody plaque-forming cells in the lung-associated lymph nodes following intratracheal immunization with sheep red blood cells. Separate groups of rats were evaluated for blastogenic responsiveness to phytohemagglutinin-P and Salmonella typhimurium antigen using spleen- and lung-associated lymph node cells. In vivo resistance was evaluated utilizing acrolein-exposed rats subsequently challenged with intravenous Listeria monocytogenes. Local pulmonary antibody responsiveness was not affected by acrolein exposure. Lymphocyte blastogenesis and resistance to Listeria challenge were not altered. Body weights and spleen weights were decreased in the 3 ppm-exposed group only. Microscopic examination of the nasal turbinates revealed acrolein-induced exfoliation, erosion, and necrosis of the respiratory epithelium as well as squamous metaplasia, however, lung histology was not affected. Thus at environmental concentrations, acrolein toxicity appeared to be confined to local nasal pathologic changes with no alterations in lung histology or immune function.

Effects of inhalation of ethylene dichloride on pulmonary defenses of mice and rats.

The effects of single or multiple inhalation exposures to ethylene dichloride (DCE) on the pulmonary defense systems of mice and rats were evaluated. Single exposures of mice to the threshold limit value of DCE (10 ppm) resulted in decreased pulmonary bactericidal activity to inhaled Klebsiella pneumoniae and increased mortality from Streptococcus zooepidemicus respiratory infection. A single exposure to 5 ppm DCE caused increased mortality from streptococcal pneumonia although bactericidal activity was not affected. Neither of these two parameters changed following single or five consecutive daily exposures to 2.5 ppm DCE. Single exposures to 10 or 100 ppm DCE did not affect mouse alveolar macrophage (AM) inhibition of the proliferation of a tumor target cell in vitro or AM in vitro phagocytosis of red blood cells. In rats, no effects were observed on pulmonary bactericidal activity. AM in vitro phagocytosis, AM cytostasis and cytolysis of tumor target cells, AM ectoenzymes, or blastogenesis of mitogen-stimulated rat T- and B-lymphocytes from lung-associated, mesenteric, and popliteal lymph nodes following single exposure to 100 or 200 ppm DCE or after twelve 5-hr exposures to 10, 20, 50, or 100 ppm DCE.