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Open Access Publications from the University of California

Scholarly Works (13 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2016)

This dissertation examines endocrine and other influences on the trafficking of immune cells. This work aimed to increase the understanding of underlying mechanisms pertaining to immune cell entry into breast milk, and prolactin’s involvement in this important physiological phenomenon. Because of observations made during this study, the work also examined various aspects of sexual dimorphism in lymph nodes and thymus.

Luminal mammary epithelial cell secretions act as chemoattractants, inducing migration of a variety of lymphocytes, phagocytes, and granulocytes. Prolactin treatment of mammary epithelial cells increases production of chemoattractants and migration of most of these cell types. This supports a role for luminal mammary epithelial cells in immune cell concentration into milk. CCL2 and CXCL1 were two chemoattractants identified responsible for a significant degree of migration of monocytes and neutrophils, respectively. In-vivo prolactin treatment increased immune cell flux through the mammary tissue and increased expression of CCL2 and CXCL1 in a mammary cell line in vitro.

Female mice have more T-cells in their popliteal lymph nodes than males, while males have a greater proportion of T-cells that are suppressive in function. This combination supports a rationale for females having a higher delayed-type hypersensitivity response in a region drained by this lymph node. The sex difference in T-cells is present prior to puberty and is to some extent dependent on Sry expression as males overexpressing Sry have even fewer T-cells. In addition, male gonadal secretions amplified the sex difference as mice aged.

Adult female mice with two X chromosomes exhibit a greater delayed-type hypersensitivity response to Candida albicans at proestrus than at metestrus or diestrus, while females with an X and a Y chromosome, the latter minus the Sry gene, did not exhibit different responses across these stages of the estrous cycle. There is therefore a combined effect of the sex chromosomal complement and hormonal changes occurring at proestrus that permits maximal response.

These results demonstrate significant interactions between the endocrine and immune systems both in regions where one might expect (the mammary gland during lactation) and in regions (the popliteal lymph node) where gene and endocrine sex effects were not anticipated.

    Cover page: Endocrine and Other Influences on the Trafficking of Immune Cells
    • Thesis
    • Peer Reviewed
    UC Riverside Electronic Theses and Dissertations (2021)

    The most common and deadly form of ovarian cancer is High-Grade Serous Ovarian Carcinoma (HGSOC). Most cases of HGSOC are now known to arise from epithelial cells of the fallopian tube and not the ovary. Lesions arise only in the fimbrial region, known as the infundibulum in the mouse. Prolactin (PRL) has been described in many studies to be a potent serum analyte in the detection of ovarian cancer. However, it is unknown whether elevated PRL initiates disease or is the result of disease. Once cancer is present, it is clear that PRL promotes cancer cell survival and migration. In human ovarian cancer cell lines, the long form (LF) of the PRL receptor (Prlr) mediates increased cell survival, proliferation and migration, whereas signaling through the short forms (SF) counteracts these effects and promotes differentiation; thus, the ratio of LF/SF Prlr is important to PRL’s end effects. To gain insight into the normal biological role of PRL in the oviduct as well as to examine PRL’s potential for disease initiation, Prlr expression and effects of elevated PRL were assessed. The ratio of LF to SF3 Prlr varied by region of the oviduct (mRNA and protein), suggesting different effects of PRL in the different oviductal segments. Interestingly, the only segment that showed changes in this ratio in response to hormones of the estrous cycle was the infundibulum. In addition, the mucosal epithelium of the infundibulum was the most responsive to acutely elevated PRL. Whole transcriptome sequencing of the infundibulum following a 7-day PRL treatment in vivo showed down regulation of genes involved in multi-ciliated cell (MCC) function. This suggests that prolonged elevated PRL reduces the function of the ciliated cells, a novel additional mechanism whereby elevated PRL negatively impacts fertility. Among additional experiments to test these hypothesis, longer term treatments with PRL will be necessary to determine whether de-differentiation of MCCs contributes to lesion formation in the infundibulum.

      Cover page: Prolactin Effect in the Murine Oviduct
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2017)
      We have previously demonstrated lactational transfer of T cell-based immunity from dam to foster pup. In the short term, a significant part of transferred immunity is passive cellular immunity. However, as time progresses, this is replaced by what we have described as maternal educational immunity such that by young adulthood, all immune cells responding to a foster dam immunogen are the product of the foster pup's thymus. To reduce confounding factors, this original demonstration used congenic/syngeneic dam and foster pup pairs. In this study, we investigated lactational transfer of immunity to Mycobacterium tuberculosis in MHC class I-mismatched animals, as well as from Th1-biased dams to Th2-biased foster pups. Using immunized C57BL/6J dams, lactational transfer to nonimmunized BALB/cJ foster pups resulted in much greater immunity than direct immunization in 5-wk-old pups (ex vivo assay of pup splenocytes). At this age, 82% of immunogen-responding cells in the pup spleen were produced through maternal educational immunity. FVB/NJ nonimmunized foster recipients had a greater number of maternal cells in the spleen and thymus but a much larger percentage was Foxp3+, resulting in equivalent immunity to direct immunization. Depletion of maternal Foxp3+ cells from pup splenocytes illustrated a substantial role for lactationally transferred dam regulatory T cells in suppression of the ex vivo response in FVB/NJ, but not BALB/cJ, recipients. We conclude that lactational transfer of immunity can cross MHC class I barriers and that Th1 immunity can be imparted to Th2-biased offspring; in some instances, it can be greater than that achieved by direct immunization.
        Cover page: Lactation-Based Maternal Educational Immunity Crosses MHC Class I Barriers and Can Impart Th1 Immunity to Th2-Biased Recipients
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2019)
        Small oligonucleotides (oligos) are increasingly being utilized as diagnostics or treatments for disease. An impediment to broader use is the ability to readily measure oligos in biological fluids. Here, we describe a very straightforward assay with detection in the sub-picomole range that does not require extraction from serum/plasma or polymerization chain reaction amplification. As a result, there are no losses or errors due to sample handling, and the assay can be used to measure oligos modified in a variety of ways that increase therapeutic efficacy. The enzyme-linked oligonucleotide hybridization assay (ELOHA) is based on competition with a detection oligo for hybridization to a capture oligo covalently linked to a solid substrate. The versatility of ELOHAs is demonstrated by application to the measurement of three oligos, including two morpholino-oligos with 3'-octaguanidine derivatization for efficient cell uptake. The third oligo is unmodified and has a DNA sequence equivalent to miR93. The assays have sensitivity as low as 0.28 pmol/sample reaction at 50% hybridization. Adding to clinical utility is the need for only a simple 96-well absorbance plate reader and the finding that neither EDTA nor heparin interferes with detection.
          Cover page: Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2008)

          Introduction

          Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult.

          Methods

          Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls.

          Results

          Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3-4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3-4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males.

          Discussion

          These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted.
            Cover page: Trans-Epithelial Immune Cell Transfer during Suckling Modulates Delayed-Type Hypersensitivity in Recipients as a Function of Gender
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2013)
            Near-infrared nanoconstructs present a potentially effective platform for site-specific and deep tissue optical imaging and phototherapy. We have engineered a polymeric nanocapsule composed of polyallylamine hydrochloride (PAH) chains cross-linked with sodium phosphate and doped with indocyanine green (ICG) toward such endeavors. The ICG-doped nanocapsules were coated covalently with polyethylene glycol (5000 daltons) through reductive amination. We administrated the constructs by tail vein injection to healthy mice. To characterize the biodistribution of the constructs, we performed in vivo quantitative fluorescence imaging and subsequently analyzed the various extracted organs. Our results suggest that encapsulation of ICG in these PEGylated constructs is an effective approach to prolong the circulation time of ICG and delay its hepatic accumulation. Increased bioavailability of ICG, due to encapsulation, offers the potential of extending the clinical applications of ICG, which are currently limited due to rapid elimination of ICG from the vasculature. Our results also indicate that PAH and ICG-doped nanocapsules (ICG-NCs) are not cytotoxic at the levels used in this study.
              Cover page: Effects of nanoencapsulation and PEGylation on biodistribution of indocyanine green in healthy mice: quantitative fluorescence imaging and analysis of organs
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2016)
              Using multiple murine foster-nursing protocols, thereby eliminating placental transfer and allowing a distinction between dam- and pup-derived cells, we show that foster nursing by an immunized dam results in development of CD8(+) T cells in nonimmunized foster pups that are specific for Ags against which the foster dam was immunized (Mycobacterium tuberculosis or Candida albicans). We have dubbed this process "maternal educational immunity" to distinguish it from passive cellular immunity. Of the variety of maternal immune cells present in milk, only T cells were detected in pup tissues. Maternal T cells, a substantial percentage of which were CD4(+)MHC class II(+), accumulated in the pup thymus and spleen during the nursing period. Further analysis of maternal cells in the pup thymus showed that a proportion was positive for maternal immunogen-specific MHC class II tetramers. To determine the outcome of Ag presentation in the thymus, the maternal or foster pup origin of immunogen-responding CD8(+) cells in foster pup spleens was assessed. Whereas ∼10% were maternally derived in the first few weeks after weaning, all immunogen-responding CD8(+) T cells were pup derived by 12 wk of age. Pup-derived immunogen-responsive CD8(+) cells persisted until at least 1 y of age. Passive cellular immunity is well accepted and has been demonstrated in the human population. In this study, we show an arguably more important role for transferred immune cells: the direction of offspring T cell development. Harnessing maternal educational immunity through prepregnancy immunization programs has potential for improvement of infant immunity.
                Cover page: Maternal Milk T Cells Drive Development of Transgenerational Th1 Immunity in Offspring Thymus
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2017)
                Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3'UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3'UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3'UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3'UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα.
                  Cover page: Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol
                  • Article
                  • Peer Reviewed
                  UC Riverside Previously Published Works (2014)
                  We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1-2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with β-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma.
                    Cover page: An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma.
                    • Article
                    • Peer Reviewed
                    UC Riverside Previously Published Works (2015)
                    Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.
                      Cover page: Antitumor Activity of a Polypyridyl Chelating Ligand: In Vitro and In Vivo Inhibition of Glioma
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