Development of an Undergraduate Cell Biology Laboratory to Assess Pigmentation and Cell Size in a Zebrafish Model of Uveal Melanoma.

This study outlines a 2-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish (Danio rerio), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type (WT) and transgenic fish [Tg(mitfa:GNAQQ209L)]. The transgenic fish express a hyperactive Gα protein, GNAQQ209L, under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins, but smaller melanophores. These results suggest there are more melanophores in the Tg(mitfa:GNAQQ209L) fish compared to the WT. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-laboratory surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.

Increase in SARS-CoV-2 RBD-Specific IgA and IgG Antibodies in Human Milk From Lactating Women Following the COVID-19 Booster Vaccination.

BACKGROUND: The United States Centers for Disease Control and Prevention recommended a third dose or booster of the Pfizer-BioNTech Comirnaty (BNT162b2) COVID-19 mRNA vaccine in September 2021 for high-risk individuals. Pregnant and high-risk lactating women were encouraged to receive the booster to obtain potential prolonged protection for themselves and their infants. RESEARCH AIM: To investigate the ability of the booster vaccine to increase IgA and IgG antibodies specific to the receptor-binding domain of the SARS-CoV-2 spike protein in human milk compared to levels pre-booster. METHODS: This was a prospective one-group study with a pretest-posttest design. Six of 12 participants were recruited prospectively. Participants were instructed to collect ≥ 2 ounces of milk in the morning at 30 days and 1-day pre-booster, and 7, 14, 21, 30, 45, and 60 days post-booster. Levels of IgA and IgG antibodies specific to the receptor-binding domain of the SARS-CoV-2 spike protein were quantified in human milk via an ELISA assay. RESULTS: We found a significant increase in anti-receptor-binding domain-specific IgA and IgG antibodies in human milk 1-2 weeks after the Pfizer-BioNTech booster and at the study endpoint (45- and 60-days post-booster). CONCLUSIONS: This suggests that the booster vaccination enhances SARS-CoV-2 specific immunity in human milk, which may be protective for infants.

Uveal melanoma driver mutations in GNAQ/11 yield numerous changes in melanocyte biology.

Uveal melanoma (UM) is the most common primary intraocular cancer and has a high incidence of metastasis, which lacks any effective treatment. Here, we present zebrafish models of UM, which are driven by melanocyte-specific expression of activating GNAQ or GNA11 alleles, GNAQ/11Q209L , the predominant initiating mutations for human UM. When combined with mutant tp53, GNAQ/11Q209L transgenics develop various melanocytic tumors, including UM, with near complete penetrance. These tumors display nuclear YAP localization and thus phenocopy human UM. We show that GNAQ/11Q209L expression induces profound melanocyte defects independent of tp53 mutation, which are apparent within 3 days of development. First, increases in melanocyte number, melanin content, and subcellular melanin distribution result in hyperpigmentation. Additionally, altered melanocyte migration, survival properties, and evasion of normal boundary cues lead to aberrant melanocyte localization and stripe patterning. Collectively, these data show that GNAQ/11Q209L is sufficient to induce numerous protumorigenic changes within melanocytes.

Downregulation of TAP1 and TAP2 in early stage breast cancer.

TAP1-TAP2 heterodimeric complexes are recognized as the transporter associated with antigen processing of major histocompatibility complex class I peptides for recognition by tumor-specific cytotoxic T lymphocytes. In this study, we investigated the immunohistochemical expression of TAP1 and TAP2 in 160 patients with breast cancer and correlated their expression levels with clinicopathologic parameters. The median age of the patient cohort was 52.5 years (range, 30-86 years). Both TAP1 and TAP2 immunohistochemical expression levels correlated significantly with breast cancer characteristics (P < .001). TAP1 expression levels were low to negative in stage I breast tumors. TAP1 and TAP2 levels were significantly higher in grade 3 tumors than low-grade (grade 1 and 2) tumors. TAP1 and TAP2 expression levels were not significantly different among different levels of HER2-expressing tumors and did not vary by estrogen and progesterone receptor status or patient age. Both TAP1 and TAP2 overexpression in breast cancer might be an indicator of an aggressive breast tumor.

Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells.

Patients with HER-2/neu-expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2-binding peptide p369-377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu-overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369-377 was processed from larger HER-2/neu-derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369-377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373-382 (SLAFLPESFD), bound HLA-A2 stronger than did p369-377. CTLs specific for p373-382 recognized both p373-382 and p369-377 complexed with HLA-A2. CTLs specific for p373-382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369-377. Conversely, CTLs specific for p369-377 recognized p373-382. Peptide p373-382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing the Cr51 release assay to the xCELLigence system.

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase release , BLT esterase activity and surface expression of CD107. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.