Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Localization and quantitation of macrophages, mast cells, and eosinophils in the developing bovine mammary gland.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is highly regulated and in mice is known to be affected by the presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. For the first time, we determined the presence, location (relative to mammary ductal structures), and changes in numbers of eosinophils, mast cells, and macrophages in prepubertal bovine mammary tissue, and evaluated the effects of age, ovariectomy, and exogenous estrogen on numbers of each cell type. Chemical stains and immunofluorescence were used to identify the 3 cell types in formalin-fixed, paraffin-embedded mammary tissue from prepubertal female calves from 3 archived tissue sets. The ontogeny tissue set included samples of mammary tissue from female calves (n=4/wk) from birth to 6 wk of age. The ovary tissue set contained samples from ovary intact and ovariectomized heifers allowing us to investigate the influence of the ovaries on immune cells in the developing mammary gland in prepubertal heifers. Nineteen animals were intact or ovariectomized 30 d before sampling; they were 90, 120, or 150 d old at the time of sampling. A third tissue set, the estrogen set, allowed us to determine the effect of exogenous estrogen on innate immune cells in the gland. Eosinophils were identified via Luna staining, mast cells by May-Grunwald Giemsa staining, and macrophages with immunofluorescence. Key findings were that more eosinophils and mast cells were observed in near versus far stroma in the ontogeny and ovary tissue sets but not estrogen. More macrophages were observed in near versus far stroma in ontogeny animals. Eosinophils were more abundant in the younger animals, and fewer macrophages tended to be observed in ovariectomized heifers as compared with intact heifers and estrogen treatment resulted in a reduction in cell numbers. In summary, we show for the first time that innate immune cells are present in prepubertal bovine mammary tissue, localization varies by immune cell type, and abundance is related to proximity of epithelial structures and physiological state. We suggest a likely role for these cells in control of bovine mammary growth and ductal development.

Oncogenic KRAS signalling promotes the Wnt/ß-catenin pathway through LRP6 in colorectal cancer.

Aberrant regulation of the Wnt/ß-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/ß-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in ß-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced ß-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished ß-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting ß-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/ß-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/ß-catenin signalings during oncogenesis.

Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants.

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01(+) rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01(+) monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.

An IL-2/Ig fusion protein influences CD4+ T lymphocytes in naive and simian immunodeficiency virus-infected Rhesus monkeys.

The T cell-stimulatory cytokine interleukin 2 (IL-2) is being evaluated as a therapeutic in the clinical settings of HIV infection and cancer. However, the clinical utility of IL-2 may be mitigated by its short in vivo half-life, toxic effects, and high production costs. We show here that an IL-2/Ig fusion protein possesses IL-2 immunostimulatory activity in vitro and a long in vivo half-life. IL-2/Ig treatment of healthy rhesus monkeys induced significant increases in CD4(+) T lymphocyte counts and expression of CD25 by these cells. Short courses of IL-2/Ig treatment of simian immunodeficiency virus (SIV)-infected rhesus monkeys in conjunction with antiretroviral drugs resulted in increased CD25 expression on T lymphocytes, and transient increases in CD4(+) T lymphocyte counts. Plasma viremia did not increase in these treated animals. Treatment of healthy or SIV-infected rhesus monkeys with a plasmid encoding the IL-2/Ig protein did not affect CD4(+) T lymphocytes. These results demonstrate that IL-2/Ig has potential utility as an immunostimulatory therapeutic.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Effect of anterior chamber-associated immune deviation (ACAID) on rat islet allograft rejection.

Anterior chamber-associated immune deviation (ACAID) is characterized by the systemic inhibition of delayed type hypersensitivity (DTH) reactions to antigens which have previously been placed into the anterior chamber of the eye. Since its initial characterization, ACAID has been elicited to a wide variety of antigens, including alloantigens, and has been shown to be due to the immune deviating effects of factors such as transforming growth factor beta (TGF-beta) in the aqueous humor on ocular antigen-presenting cells (APCs). ACAID can also be induced by injecting animals with nonocular APCs, such as peritoneal exudate cells (PECs), which have been precultured with TGF-beta and antigen in vitro. The objective of this study was to determine whether alloantigenic ACAID can be effective in preventing the rejection of rat islet allografts. The notion that islet allograft rejection can be inhibited by ACAID stems from an early study showing an ACAID-induced delay in the rejection of skin grafts. Furthermore, the immune cells mediating a DTH reaction are similar to those implicated in islet allograft rejection, suggesting that they, too, may be subject to inhibition by ACAID. Our results showed that in spite of successful ACAID induction to islet allografts, recipient rats consistently rejected their grafts. Cytotoxic T cell activity (which is not inhibited by ACAID) directed against donor alloantigens was high in these animals and may have accounted, in part, for graft failure.