Michael L. Misfeldt

Sinclair miniature swine: an animal model of human melanoma.

Sinclair swine display cutaneous melanoma lesions and develop a generalized depigmentation subsequent to tumor regression. Sinclair swine represent a valuable animal model to study the factors influencing the development of melanoma and also the factors which lead to the development of vitiligo. Therefore, information obtained in studies of Sinclair swine should facilitate our understanding of the mechanisms by which melanoma and vitiligo develop and provide us with possible therapeutic treatments for these human diseases.

17beta-estradiol suppresses TLR3-induced cytokine and chemokine production in endometrial epithelial cells

BackgroundThe human endometrium is an important site for contact between the host and pathogens ascending the reproductive tract, and thus plays an important role in female reproductive tract immunity. Previous work in our laboratory has suggested that Toll-like receptors (TLRs) are involved in endometrial epithelial recognition of pathogens and that ligation of endometrial TLRs results in the production of cytokines and chemokines important for both immune and reproductive functions of the endometrium. We have also demonstrated cyclic regulation of TLR3 mRNA and protein expression in human endometrium, suggesting that steroid hormones might play a role in the expression and function of TLR3. In this study, the effects of 17beta-estradiol (E2) and progesterone (P) on TLR3 expression and function in endometrial cell lines were investigated.MethodsEndometrial epithelial cell lines were cultured and examined for the presence of TLR3 and hormone receptors by endpoint RT-PCR. For hormonal studies, cells were pre-treated with ethanol vehicle, 10^(-8) M E2, and/or 10^(-7) M P. For antagonist assays, cells were treated with the ER antagonist, ICI 182, 780, or the PR antagonist, RU486, for two hours prior to treatment with hormones. Following hormone or hormone/antagonist pre-treatment, cells were stimulated with vehicle, the synthetic TLR3 ligand, polyinosinic-polycytidylic acid (Poly I:C), a negative dsDNA control, or a positive control. Cytokine and chemokine production post-stimulation was measured by ELISA. The effects of E2 and P on TLR3 mRNA and protein expression were measured using Real Time RT-PCR and FACS analysis, respectively.ResultsStimulation of TLR3-expressing cells with the synthetic TLR3 ligand, Poly I:C, resulted in the production of cytokines and chemokines important for endometrial function and regulation. Suppression of Poly I:C-induced cytokine and chemokine production by cells treated with 10^(-8) M E2, but not cells treated with 10^(-7) M P, was observed in endometrial epithelial cell lines expressing TLR3 and estrogen receptor alpha (ERalpha). The effects of E2 were not observed on cells which did not express ERalpha or in cells pre-treated with the ER antagonist, ICI 182, 780. Treatment with E2 did not affect TLR3 mRNA or protein expression. However, treatment with E2 did suppress cytokine and chemokine production resulting from TLR3 stimulation with Poly I:C, suggesting that E2 modulates TLR3 function.ConclusionThe data presented in this study are the first indication that E2 can markedly alter the innate immune response to dsRNA, providing a previously unreported process by which E2 can alter immune responses.

Analyses of monoclonal antibodies reactive with porcine CD44 and CD45

Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.

The superantigen Pseudomonas exotoxin a requires additional functions from accessory cells for T lymphocyte proliferation

We have examined the functions required of accessory cells (AC) for murine thymocyte proliferation induced by Pseudomonas exotoxin A (PE) and have compared these functions to those required of a known superantigen, staphylococcal enterotoxin B (SEB). We demonstrate that PE, like SEB, preferentially stimulates PNA+ thymocytes expressing a specific V beta element within the T cell receptor. However, PE requires functions from AC that are distinct from those required by SEB. AC treated with paraformaldehyde (PCHO) prior to stimulation supported thymocyte proliferation induced by SEB but not PE. However, when AC were treated with PCHO subsequent to stimulation with PE, thymocyte proliferation was observed, which suggests that PE requires antigen processing in addition to presentation. Furthermore, treatment of AC with lysosomotropic agents abrogated thymocyte proliferation induced by PE but not SEB. Antibodies to MHC class II molecules inhibited thymocyte proliferation induced by both PE and SEB. In addition, we observed that interleukin 1 alpha (IL-1 alpha) participated in the proliferation of thymocytes induced by PE but not SEB. Thus, our data indicate that PE is a unique microbial superantigen that requires additional AC functions for T lymphocyte proliferation.

Endocrine and thermoregulatory responses to acute thermal exposures in 6-month-old pigs reared in different neonatal environments

1. 1. Endocrine and thermoregulatory responses to acute heat (34°C) and cold (10°C) exposures were determined in eight pigs at 6 months of age. Half of the pigs had been reared in a cycling upper thermal environment (27–32°C) for the first 28 days of life, while the others had been reared in a lower thermal environment (21°C). 2. 2. Concentrations of cortisol increased significantly during both acute heat and cold thermal exposures (P = 0.0001) although the response was greater in the heat than in the cold (P = 0.003). A prolactin response occurred during acute heat exposure (P = 0.004). Growth hormone secretion increased during acute cold exposure (P = 0.001). There was a strong tendency for increased epinephrine secretion during both acute heat and cold exposures (P = 0.06). No significant effects of either exposure were found on plasma norepinephrine (P = 0.9), or triiodothyronine (P = 0.11). 3. 3. Neonatal environment did not affect daily core body temperature (Tc) before acute heat or cold exposures, but did alter amplitude of the 24-h Tc cycle. The amplitude was significantly greater in animals reared in the lower thermal environment (P < 0.001). 4. 4. Acute heat exposure resulted in significant increases in Tc (P < 0.001) and heat production (P < 0.01). Neonatal thermal environment had no significant effect on Tc and heat production responses to acute heat and cold thermal exposures.

Host environment as a modulating factor of swine natural killer cell activity

The large granular lymphocyte (LGL) population includes such heterologous effector cells as the natural killer (NK), lymphokine activated killer (LAK), antibody dependent cellular cytotoxic (ADCC) and non-MHC restricted T cells. These LGL subpopulations have all been associated with NK activity. In some species, enhanced NK activity is correlated with exposure to viral, bacterial and parasitic agents. Consequently, the host environment could serve as a modulatory factor of NK activity in laboratory animals. During our investigation of tumor regression in melanoma swine, we observed marked differences in the NK activity of peripheral blood lymphocytes collected from two separate groups of Sinclair melanoma miniature swine maintained under different conditions. Group A pigs were vaccinated and extensively treated for endo- and ectoparasites while group B swine were not. In addition, chronic exposure to infectious and parasitic diseases have been documented in the group B swine. Peripheral blood NK activity was assessed by standard in vitro 4-h chromium release assays. The NK activity of group B swine was markedly exaggerated when compared to group A swine. Thus, the significance of NK activity may be distorted as a result of the modulating effect of pathogen exposure.

Isolation and characterization of γδ T lymphocyte cell lines from Sinclair swine peripheral blood

Abstract Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2−, CD4−, CD8−, and slg−, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the γδ T cell receptor (TCR) heterodimer, swine γδ T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of γδ TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the Cδ chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR γ chain gene and express mRNA from the TCR γ and δ chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine γδ T lymphocyte cell lines which should enable us to enhance our understanding of the role of γδ T lymphocytes in the porcine immune system.

Toll-like receptors in tonsillar epithelial cells

OBJECTIVE The Waldeyers ring, comprised of the nasopharyngeal tonsil, the paired tubal tonsils, the paired palatine tonsils, and the lingual tonsil, is arranged in a circular orientation around the wall of the throat. The location of the palatine tonsils, specifically, enables these structures to come in direct contact with potentially harmful inhaled and ingested material that exist in their native form since digestive enzymes are not present in the oral cavity. Thus, the tonsillar epithelium must not only serve a protective role but it must also function in an antigen-sampling role. Previous studies involving the tissues of the Waldeyers ring have been focused on the adaptive immune system, with little consideration toward the innate immune system. Studies have demonstrated that the tonsils are capable of producing proinflammatory and antiviral cytokines and chemokines. In addition, other studies have highlighted the importance of epithelial cells in this response. Therefore, we postulate that toll-like receptors (TLRs), which recognize components of pathogenic organisms, may play a key role in the innate immune response in tonsillar epithelial cells. TLRs are innate pattern recognition receptors, which produce proinflammatory cytokines and chemokines upon ligation. In this study, we examine the expression and function of TLRs in the tonsillar epithelial cell lines, UT-SCC-60A and UT-SCC-60B. Additionally, we demonstrate successful isolation of primary tonsillar epithelial cells and examine TLR expression in these cells. METHODS We utilized endpoint RT-PCR, real time RT-PCR, and flow cytometric analysis to determine TLR expression. To assess TLR function, cells were stimulated with TLR ligands and supernatants were assayed for secretion of cytokines. RESULTS UT-SCC-60A and UTSCC-60B express TLR mRNA and TLR protein, and the observed responses to the TLR ligands, Pam3Cys and Poly I:C suggest that TLR2 and TLR3 are functional in these cells. Additionally, primary tonsillar epithelial cells express TLRs. CONCLUSIONS TLRs are expressed in human tonsillar epithelial cells and may play a vital role in the immunological outcomes in this tissue.

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.

Selective activation of murine Vβ8.2 bearing T cells by Pseudomonas exotoxin A

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.