Benny L. Blaylock

Nonspecific stimulation of the maternal immune system. I. Effects on teratogen-induced fetal malformations

BACKGROUND Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freunds complete adjuvant (FCA). RESULTS Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Single-dose topical exposure to the pyrethroid insecticide, permethrin in C57BL/6N mice: effects on thymus and spleen

Immunomodulatory effects of single topical exposure to permethrin were evaluated in 5-week-old female C57BL/6N mice. Mice exposed to 5-25 microl permethrin (equivalent to 220-1100 mg/kg body weight) on shaved interscapular skin were evaluated for altered body weight; splenic and thymic organ weight and cellularity; thymocyte cell surface expression, cellular apoptosis; splenic macrophage phagocytosis and hydrogen peroxide production; splenic B cell antibody production and T cell cytolytic activity; and mitogen-induced proliferation of splenocytes and thymocytes after in vivo or in vitro permethrin exposure. Topical permethrin application (25 microl) caused 32% inhibition of splenic T cell proliferation; in vitro exposure to permethrin also diminished splenocyte proliferation by 72% at 25 microM and 86% at 100 microM. permethrin did not appear to affect other leukocyte functional assays. Dose-related decreases in thymic cellularity of 52 and 80% were seen in mice exposed to 15 and 25 microl permethrin, respectively. Apoptosis was significantly increased in CD4(-)8(-) and CD4(-)8(+) thymocytes, and the CD4(+)CD8(+) thymocyte subpopulation was most severely diminished, suggesting possible chemical-induced apoptotic mechanism of thymic atrophy. Permethrin also caused splenic hypocellularity by 31% at 15 microl, and by 50% at 25 microl, an effect that may relate to inhibited proliferation or reduced seeding from the hypocellular thymus.

Topical permethrin exposure inhibits antibody production and macrophage function in C57Bl/6N mice.

Permethrin was applied to the shaved dorsal interscapular region of C57Bl/6N mice at doses of 0.5, 1.5 or 5.0 microl/day. These doses corresponded to approximately 22-220 mg/kg/day topical insecticide. Mice were exposed to permethrin in this manner daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. The splenic macrophage chemiluminescent response was depressed in a dose-dependent manner at 2 and 10 days post-exposure to permethrin. Phagocytic ability of macrophages was not inhibited. Antibody production as shown by plaque-forming cell (PFC) assay decreased significantly after 10 consecutive days of exposure to permethrin. These data indicate that topical permethrin exposure may produce systemic immune effects.

Hematopoietic alterations after exposure to T-2 mycotoxin

Adult mice were exposed by oral gavage to 0.75, 1.25, or 1.75 mg/kg body weight T-2 mycotoxin for 5 consecutive days. Thymic atrophy on the 2nd day following cessation of dosing was profound, and was characterized by significant decreases in the total number of cells within all phenotypes defined by CD4 and CD8 cell-surface antigen expression. Further, the distribution of thymocytes within these phenotypes was significantly altered. Increased percentages of CD4-8- (DN) and decreased percentages of CD4+8+ (DP) cells in thymuses from treated animals suggested that T-2 toxin may inhibit thymocyte maturation. In addition to thymus, the bone marrow of treated animals showed a highly significant hypocellularity, indicating that this hematopoietic compartment may also be targeted by T-2 toxin. A trend toward reduced splenic cellularity was additionally observed in exposed animals, but failed to reach significance. A significant decrease in the total number of both B and T-lymphocytes present within the spleen was observed, however. These data, taken together, indicate that effects at multiple hematopoietic compartments involved in the production of T-lymphocytes may contribute to the peripheral T-cell lymphocytopenia and T-cell mediated immunosuppression produced by T-2 toxin.

The opposite effects of doxorubicin on bone marrow stem cells versus breast cancer stem cells depend on glucosylceramide synthase.

Myelosuppression and drug resistance are common adverse effects in cancer patients with chemotherapy, and those severely limit the therapeutic efficacy and lead treatment failure. It is unclear by which cellular mechanism anticancer drugs suppress bone marrow, while drug-resistant tumors survive. We report that due to the difference of glucosylceramide synthase (GCS), catalyzing ceramide glycosylation, doxorubicin (Dox) eliminates bone marrow stem cells (BMSCs) and expands breast cancer stem cells (BCSCs). It was found that Dox decreased the numbers of BMSCs (ABCG2(+)) and the sphere formation in a dose-dependent fashion in isolated bone marrow cells. In tumor-bearing mice, Dox treatments (5mg/kg, 6 days) decreased the numbers of BMSCs and white blood cells; conversely, those treatments increased the numbers of BCSCs (CD24(-)/CD44(+)/ESA(+)) more than threefold in the same mice. Furthermore, therapeutic-dose of Dox (1mg/kg/week, 42 days) decreased the numbers of BMSCs while it increased BCSCs in vivo. Breast cancer cells, rather than bone marrow cells, highly expressed GCS, which was induced by Dox and correlated with BCSC pluripotency. These results indicate that Dox may have opposite effects, suppressing BMSCs versus expanding BCSCs, and GCS is one determinant of the differentiated responsiveness of bone marrow and cancer cells.

Implementing a Referral to Telephone Tobacco Cessation Services in Louisiana Community Pharmacies: A Pilot Study

Background: With one of the highest rates of tobacco dependence in the nation, Louisiana has been searching for economical and effective methods for assisting patients in cessation efforts. Community pharmacists are in an excellent position to promote tobacco cessation due to their availability to patients. The “Ask-Advise-Refer” model is a short intervention in which patients desiring to quit smoking are referred to free tobacco cessation telephone counseling services. Objective: To evaluate the implementation of the Ask-Advise-Refer model in a sample of Louisiana pharmacies and identify barriers experienced by pharmacists when identifying and referring appropriate patients. Methods: Nine pharmacists from across the state implemented the Ask-Advise-Refer model in their community pharmacies. Each pharmacist submitted a weekly tally sheet consisting oí number of patients asked about tobacco dependence, number of patients not ready to quit, number referred to tobacco cessation telephone counseling, number enrolled in study, and amount of lime involved with interventions. Additionally, participating pharmacists completed a self-administered survey at the completion of the pilot study to determine opinions on barriers to widespread implementation of the program. Results: Over a 6-month period, the 9 pharmacists asked 5429 patients about tobacco dependence. Of the 657 self-identified tobacco-dependent patients, 478 (72.8%) were not ready to quit, and 179 (27.2%) indicated that they were ready to quit tobacco in the next 30 days. Of the patients ready to quit, 169 (94.4%) were referred to telephone counseling services to assist in their cessation efforts. Conclusions: Louisiana community pharmacists have the ability to screen and identify tobacco-dependent patients ready to quit tobacco use, but barriers exist that prevent a large number of these patients from being referred to available, free cessation counseling,

Topical exposure to chlordane reduces the contact hypersensitivity response to oxazolone in BALB/c mice

Previous studies have shown that prenatal exposure to the organochlorine pesticide chlordane significantly decreases the ear swelling response to the contact allergen oxazolone in BALB/c mice. Alterations of macrophage function in the efferent arm of the contact hypersensitivity response have also been reported. In the current study, chlordane was applied topically and the effects of oxazolone-induced contact hypersensitivity were determined. Initially, the reduction in oxazolone-induced ear swelling in topically-exposed female BALB/c mice was compared to 30-day-old BALB/c female mice exposed prenatally to chlordane. Prenatal chlordane exposure induced a 36% reduction in ear swelling compared to a 60% reduction following topical treatment at the challenge phase. Topically-applied chlordane also reduced the oxazolone-induced ear swelling by 40% when applied at sensitization. When applied at both sensitization and challenge, ear swelling was reduced by 71%. In a time-course study, it was determined that chlordane must be applied at the time of sensitization, challenge or both or within 1 h post-challenge to significantly reduce ear swelling. A dose-response study showed that the lowest concentration of chlordane resulting in a significantly reduced ear swelling response was 20 micrograms per ear.

Cis-urocanic acid increases immunotoxicity and lethality of dermally administered permethrin in C57BL/6N mice

Immunomodulatory effects of a single topical permethrin exposure, 5-day exposure to cis-urocanic acid (c UCA), or a combination of the two chemicals were evaluated in 4- to 5-week-old female C57BL/6N mice. Permethrin alone decreased thymic weight and cellularity. Although c UCA alone did not affect thymic end points, coexposure to topical permethrin and c UCA exacerbated the thymolytic effects of permethrin. The single topical dose of permethrin also depressed several immune responses in isolated splenic leukocytes. This included splenic T-cell proliferative response to mitogen, splenic macrophage hydrogen peroxide production, and splenic B lymphocyte-specific antibody production. Unlike the effect of coexposure to these agents on thymic end points, c UCA did not exacerbate permethrins adverse effect on any of the splenic end points examined. These results appear to suggest divergent mechanisms by which these compounds affect precursor and functionally mature T cells. At the doses used in this study, permethrin caused neurotoxic effects, including lethality, in a portion of the mice. For undetermined reasons, c UCA significantly increased the rate of lethality caused by permethrin. Although the permethrin doses used in this study exceed that typically used in human medicine, these results raise some concerns about the possibility that sunlight, via c UCA, may increase the risk of adverse central nervous system and immune effects caused by permethrin alone.

Topical Permethrin Exposure Causes Thymic Atrophy and Persistent Inhibition of the Contact Hypersensitivity Response in C57BI/6 Mice

Permethrin was applied to the shaved dorsal interscapular region of female C57B1/6 mice at doses of 0.5 or 1.5 θ 1/day in corn oil and neat 5.0 θ 1/day. These doses corresponded to approximately 22, 66, and 220 mg/kg/day topical permethrin. Mice were exposed in this manner either daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. Body weight was not affected by any of the treatment regimens. However, thymic weight was decreased and splenic weight was increased by 1.5 or 5.0 θ1 permethrin/day, 2 days after termination of 10 consecutive days of topical chemical exposure. Cell surface antigen expression did not change in any treatment group on thymocytes (CD4, CD8), splenocytes (CD45R, Thy 1.2), or bone marrow cells (CD45, CD45R). A persistent, dose-related inhibition of the contact hypersensitivity (CH) response occurred in mice at all exposure levels of permethrin tested.

Experimental Mathematical Validation of a Novel Concept of Extended 2-Butoxyethanol Autoprotection.

Prior administration of a moderately hemolytic dose of 2-butoxyethanol (2-BE) in rats leads to autoprotection against the lethal effects of high doses. Digavalli and Mehendale (Arch. Toxicol. 69:526–532;1995) showed that this autoprotection was due to recovery from the prior episode of hemolysis resulting in a higher proportion of young red blood cells (RBCs), which are more resilient to 2-BE. The objective of this research was to investigate the hypothesis that autoprotection can be entirely explained in terms of such changes in age composition of the RBC population. A simple simulation model was developed to provide predictions of the effect of various 2-BE dosage regimes, which were then experimentally verified. Some model predictions were confirmed by the experiments, but others were distinctly off the mark. The longer sequences (two or three successive episodes) in particular showed unexpected erythropoietic responses. This was further investigated in additional experiments, which also looked at reticulocyte counts and serum levels of erythropoietin (Epo). These showed that the release of reticulocytes following a 2-BE challenge is considerably faster than one would expect from the normal processing time in the bone marrow, and also becomes stronger at each successive challenge, resulting in remarkably high levels of reticulocytosis. It is concluded that changes in age composition of the RBCs cannot fully explain the time course of the hematocrit during consecutive administration of several doses of 2-BE, and that other mechanisms must play an important role as well. The results seem to indicate that after the hematocrit has recovered a buffer of (almost mature) reticulocytes remains available in the bone marrow for several days, which can be released almost immediately when another decrease in hematocrit is evoked.