Characterizing Differences in Thymic Function in Women With and Without Vulvodynia: A Community-Based Study.

OBJECTIVE: The aim of the study was to evaluate the association between vulvodynia and thymic function. MATERIALS AND METHODS: In this case-control study of 200 clinically confirmed cases of vulvodynia and 205 general population controls residing in the Minneapolis/Saint Paul metropolitan area, we used DNA extracted from whole blood to measure levels of signal joint T-cell receptor excision circles (sjTRECs), a measure of thymic output. We used logistic regression to evaluate the association between vulvodynia and thymic function. RESULTS: In 405 participants (aged 18-40 years), we observed an association between decreasing thymic function and increasing age. Women with vulvodynia had a steeper decline in sjTREC values across age categories compared with women without vulvodynia. In addition, at younger ages, women with vulvodynia had higher sjTREC values compared with women without vulvodynia. In older women, those with vulvodynia had lower sjTREC than those without vulvodynia. When accounting for recency of vulvar pain onset, women with a shorter time since pain onset had higher thymic function compared with women with a longer time since vulvar pain onset. CONCLUSIONS: These findings suggest that at younger ages, women with vulvodynia have higher thymic output and a more precipitous decline of thymic function than those without vulvodynia. It also seems that a strong immune inflammatory response is present proximate to the onset of vulvar pain and may wane subsequently over time.

Tetrahydrocannabinol Reduces Hapten-Driven Mast Cell Accumulation and Persistent Tactile Sensitivity in Mouse Model of Allergen-Provoked Localized Vulvodynia.

Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. An increase in numbers of vulvar mast cells often accompanies a clinical diagnosis of vulvodynia and a history of allergies amplifies the risk of developing this condition. We previously showed that repeated exposures to oxazolone dissolved in ethanol on the labiar skin of mice led to persistent genital sensitivity to pressure and a sustained increase in labiar mast cells. Here we sensitized female mice to the hapten dinitrofluorobenzene (DNFB) dissolved in saline on their flanks, and subsequently challenged them with the same hapten or saline vehicle alone for ten consecutive days either on labiar skin or in the vaginal canal. We evaluated tactile ano-genital sensitivity, and tissue inflammation at serial timepoints. DNFB-challenged mice developed significant, persistent tactile sensitivity. Allergic sites showed mast cell accumulation, infiltration of resident memory CD8+CD103+ T cells, early, localized increases in eosinophils and neutrophils, and sustained elevation of serum Immunoglobulin E (IgE). Therapeutic intra-vaginal administration of Δ9-tetrahydrocannabinol (THC) reduced mast cell accumulation and tactile sensitivity. Mast cell-targeted therapeutic strategies may therefore provide new ways to manage and treat vulvar pain potentially instigated by repeated allergenic exposures.

Repeated Vaginal Exposures to the Common Cosmetic and Household Preservative Methylisothiazolinone Induce Persistent, Mast Cell-Dependent Genital Pain in ND4 Mice.

A history of allergies doubles the risk of vulvodynia-a chronic pain condition of unknown etiology often accompanied by increases in numbers of vulvar mast cells. We previously established the biological plausibility of this relationship in mouse models where repeated exposures to the allergens oxazolone or dinitrofluorobenzene on the labiar skin or inside the vaginal canal of ND4 Swiss Webster outbred mice led to persistent tactile sensitivity and local increases in mast cells. In these models, depletion of mast cells alleviated pain. While exposure to cleaning chemicals has been connected to elevated vulvodynia risk, no single agent has been linked to adverse outcomes. We sensitized female mice to methylisothiazolinone (MI)-a biocide preservative ubiquitous in cosmetics and cleaners-dissolved in saline on their flanks, and subsequently challenged them with MI or saline for ten consecutive days in the vaginal canal. MI-challenged mice developed persistent tactile sensitivity, increased vaginal mast cells and eosinophils, and had higher serum Immunoglobulin E. Therapeutic and preventive intra-vaginal administration of Δ9-tetrahydrocannabinol reduced mast cell accumulation and tactile sensitivity. MI is known to cause skin and airway irritation in humans, and here we provide the first pre-clinical evidence that repeated MI exposures can also provoke allergy-driven genital pain.

Semaphorin 4A is dynamically regulated during thymocyte development in mice.

Semaphorins are important regulators of peripheral T and B-cell mediated immune responses in mice and humans. Modulatory roles of semaphorins in T cell development are also being characterized. We carefully analyzed the gene expression and protein levels of semaphorins 4A, 4D, and 7A at various developmental stages of T cell maturation in the thymus of C57BL/6 mice. Sema7a was expressed at very low levels, while Sema4d was abundant at all developmental stages of mouse thymocytes. We found the most interesting pattern of gene regulation and protein localization for semaphorin 4A. Both semaphorin 4A mRNA and protein were clearly detected on the earliest progenitors and were downregulated through thymic development. SEMA4A protein also showed a distinct cortico-medullary pattern of localization. Our findings contribute to an understanding of the complex roles played by semaphorins in the network of spatially and temporally regulated cues underpinning T cell development in the thymus.

Immune mechanisms in vulvodynia: key roles for mast cells and fibroblasts.

Vulvodynia is a debilitating condition characterized by painful sensitivity to touch and pressure in the vestibular tissue surrounding the vaginal opening. It is often a "diagnosis of exclusion" of idiopathic pain made in the absence of visible inflammation or injury. However, the association between increased vulvodynia risk and a history of yeast infections and skin allergies has led researchers to explore whether immune mechanisms of dysregulated inflammation might underlie the pathophysiology of this chronic pain condition. Here we synthesize epidemiological investigations, clinical biopsies and primary cell culture studies, and mechanistic insights from several pre-clinical models of vulvar pain. Taken together, these findings suggest that altered inflammatory responses of tissue fibroblasts, and other immune changes in the genital tissues, potentially driven by the accumulation of mast cells may be key to the development of chronic vulvar pain. The association of increased numbers and function of mast cells with a wide variety of chronic pain conditions lends credence to their involvement in vulvodynia pathology and underscores their potential as an immune biomarker for chronic pain. Alongside mast cells, neutrophils, macrophages, and numerous inflammatory cytokines and mediators are associated with chronic pain suggesting immune-targeted approaches including the therapeutic administration of endogenous anti-inflammatory compounds could provide much needed new ways to treat, manage, and control the growing global pandemic of chronic pain.

The C-C chemokine receptor 7: An immune molecule that modulates central nervous system function in homeostasis and disease.

The interaction between the central nervous system (CNS) and the peripheral immune system is key for brain function in homeostasis and disease. Recent studies have revealed that the C-C chemokine receptor 7 (CCR7) is expressed in both CNS resident cells and peripheral immune cells, and plays an important role in regulating behavior in homeostasis and neuroinflammation in disease. This review integrates studies examining the role of CCR7 in CNS resident and peripheral immune cells in homeostasis and disease, as well as the pathways of peripheral immune cell migration in and out of the brain via CCR7. A special emphasis is placed on the CCR7-dependent migration of peripheral immune cells into the recently discovered meningeal lymphatic vessels surrounding the brain and nasal lymphatics, its migration into cervical lymph nodes, and the implications that this migration might have for CNS function.

Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice.

Mast cells mediate allergies, hypersensitivities, host defense, and venom neutralization. An area of recent interest is the contribution of mast cells to inflammatory pain. Here we found that specific, local activation of mast cells produced plantar hyperalgesia in mice. Basic secretagogue compound 48/80 induced plantar mast cell degranulation accompanied by thermal hyperalgesia, tissue edema, and neutrophil influx in the hindpaws of ND4 Swiss mice. Blocking mast cell degranulation, neutrophil extravasation, and histamine signaling abrogated these responses. Compound 48/80 also produced edema, pain, and neutrophil influx in WT C57BL/6 but not in genetically mast cell-deficient C57BL/6-Kit(W-sh)(/)(W-sh) mice. These responses were restored following plantar reconstitution with bone marrow-derived cultured mast cells.

Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.

Endothelin-1 (ET-1) is a 21-amino-acid peptide, derived from vascular endothelial cells, with potent vasoconstrictor activity. ET-1 has been implicated in diverse physiological or pathological processes, including the vascular changes associated with sepsis. However, the factors that regulate ET-1-associated toxicity during bacterial infections, or in other settings, are not fully understood. Both the pathology associated with certain allergic and autoimmune disorders, and optimal host defence against bacterial and parasitic infections are mediated by mast cells. In vitro, mast cells can produce ET-1 (ref. 11), undergo ET-1-dependent and endothelin-A receptor (ET(A))-dependent activation, and release proteases that degrade ET-1 (ref. 14). Although the potential relationships between mast cells and the ET-1 system thus may be complex, the importance of interactions between ET-1 and mast cells in vivo is obscure. Here we show that ET(A)-dependent mast-cell activation can diminish both ET-1 levels and ET-1-induced pathology in vivo, and also can contribute to optimal survival during acute bacterial peritonitis. These findings identify a new biological function for mast cells: promotion of homeostasis by limiting the toxicity associated with an endogenous mediator.

Teaching Immunology as a Liberal Art.

A complex, rapidly evolving biomedical field that is of critical relevance to human health and well-being, immunology provides important and substantive opportunities to practice and teach the central tenets of a liberal arts curriculum.

Generating primary murine vaginal fibroblast cell lines.

Primary human vulvovaginal fibroblast cell lines are useful for studying biological mechanisms underlying genital pain, pelvic organ prolapse, and the spread of sexually transmitted infections. However, the vaginal biopsies necessary for establishing these cell lines are invasive and relatively difficult to obtain. Primary mouse fibroblast cell lines derived from pre-clinical animal models of these conditions can be used for better controlled experiments that can help us dissect disease mechanisms. To our knowledge, there are no published protocols for establishing primary murine vaginal fibroblast cell lines to date. Here, we describe a protocol for the establishment of murine vaginal fibroblast cell lines via enzymatic digestion of vaginal canal tissue. Cell lines generated using this method can be used for in vitro studies of these important structural cells in a variety of pre-clinical mouse models; such studies are required to identify and characterize relevant regulatory and therapeutic targets in a wide array of diseases of interest. As shown in our representative data, this protocol yields viable cell lines from ND4 Swiss outbred mice. These cells bear surface markers characteristic of fibroblasts and are capable of producing inflammatory cytokines in response to treatment with bacterial and yeast antigens in vitro.

Repeated dermal application of the common preservative methylisothiazolinone triggers local inflammation, T cell influx, and prolonged mast cell-dependent tactile sensitivity in mice.

Occupational exposure to toxic chemicals increases the risk of developing localized provoked vulvodynia-a prevalent, yet poorly understood, chronic condition characterized by sensitivity to touch and pressure, and accumulation of mast cells in painful tissues. Here, we topically sensitized female ND4 Swiss mice to the common household and industrial preservative methylisothiazolinone (MI) and subsequently challenged them daily with MI or acetone and olive oil vehicle on the labiar skin. MI-challenged mice developed significant, persistent tactile sensitivity and long-lasting local accumulation of mast cells alongside early, transient increases in CD4+ and CD8+ T cells, eosinophils, neutrophils, and increases in pro-inflammatory cytokines. Therapeutic administration of imatinib, a c-Kit inhibitor known to inhibit mast cell survival, led to reduced mast cell accumulation and alleviated tactile genital pain. We provide the first pre-clinical evidence of dermal MI-induced mast-cell dependent pain and lay the groundwork for detailed understanding of these intersections between MI-driven immunomodulation and chronic pain.

Ready, Cell, Go! Guided Movement Exercises in an Introductory Cell Biology Course.

Cells and dance are each dynamic manifestations of energy, shape, time, and space. Here we present a novel application of movement learning in cell biology education. "Ready, Cell, Go!" is a set of movement exercises for introductory cell biology students designed to teach concepts of fluidity, crowding, and chaos. These aspects of cells are difficult to glean from two-dimensional illustrations in textbooks or animations where necessary simplification abstracts processes from their full cellular context. Forty-four undergraduate biology students were guided to move using three sets of cues in a dance studio setting where each exercise aimed to experientially highlight and deepen understanding of a different aspect of cellular structure and function. Students described their experiences and personal learning outcomes in written reflections. The movement-based exercises we describe provided a means of discovery, inquiry, and interest for introductory cell biology students and serve as a template to teach other central concepts in cell biology.

IL-3 is required for increases in blood basophils in nematode infection in mice and can enhance IgE-dependent IL-4 production by basophils in vitro.

Basophils represent potential effector and immunoregulatory cells, as well as a potential source of IL-4, during the immune response elicited by infection with the nematode Nippostrongylus brasiliensis (N.b.), and in other settings. However, the factors which regulate the numbers of blood basophils in mice, or the ability of these cells to produce IL-4, are not fully understood. We found that infection of mice with the nematodes N.b. or Strongyloides venezuelensis (S.v.) induced substantial increases in the numbers of blood basophils (to as high as 18% of circulating blood leukocytes). Experiments in IL-3-/- vs IL-3+/+ mice, and in IL-3-treated IL-3-/- mice, showed that essentially all of the increases in blood or bone marrow basophils during N.b. or S.v. infection were IL-3 dependent. Many of the blood, bone marrow or liver-derived basophils from IL-3-/- or IL-3+/+ mice expressed intracellular IL-4 upon stimulation with anti-IgE in vitro. However, after incubation of the cells with exogenous IgE in vitro, blood- or liver-derived basophils from IL-3+/+ mice exhibited higher levels of intracellular IL-4 after stimulation with anti-IgE than did basophils derived from IL-3-/- mice. Thus, IL-3 is a major regulator of the marked increases in blood basophil levels observed during infection of mice with N.b. or S.v. and also can enhance levels of intracellular IL-4 upon activation of basophils with anti-IgE in vitro.

Recurrent Yeast Infections and Vulvodynia: Can We Believe Associations Based on Self-Reported Data?

OBJECTIVE: We determined whether self-reported new or recurrent yeast infections were a risk factor for and/or consequence of vulvodynia and then determined the extent to which various levels of misclassification of self-reported yeast infections influenced these results. MATERIALS AND METHODS: In this case-control study we retrospectively assessed self-reported new and recurrent yeast infections prior and subsequent to first vulvar pain onset among 216 clinically confirmed cases and during a similar time period for 224 general population controls. RESULTS: A history of >10 yeast infections before vulvodynia onset was strongly but imprecisely associated with currently diagnosed vulvodynia after adjustment for age, age at first intercourse, and history of urinary tract infections [adjusted odds ratio = 5.5, 95% confidence interval (CI) 1.7-17.8]. Likewise, a history of vulvodynia was associated with a twofold risk of subsequent new or recurrent onset of yeast infections after adjustment for age, age at first intercourse, and history of yeast infections before vulvodynia onset (comparable time period among controls, 95% CI 1.5-2.9). Bias analyses showed that our observed associations were an underestimation of the true association when nondifferential misclassification of self-reported yeast infections and certain differential misclassification scenarios were present. However, if women with vulvodynia more frequently misreported having them when they truly did not, our observed associations were an overestimate of the truth. CONCLUSIONS: There appears to be a positive relationship between yeast infections preceding and following the diagnosis of vulvodynia, but this relationship varies from strong to nonexistent depending on the relative accuracy of the recalled diagnosis of yeast infections among cases and controls. To better understand the bidirectional associations between yeast infections and vulvodynia, future validation studies are needed to determine the extent to which misclassification of self-reported yeast infections differs between women with and without vulvodynia.

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia.

BACKGROUND: Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. Epidemiologic studies associate the risk of vulvodynia with a history of atopic disease. We used an established model of hapten-driven contact hypersensitivity to investigate the underlying mechanisms of allergy-provoked prolonged sensitivity to pressure. METHODS: We sensitized female ND4 Swiss mice to the hapten oxazolone on their flanks, and subsequently challenged them four days later with oxazolone or vehicle for ten consecutive days on the labia. We evaluated labiar sensitivity to touch, local mast cell accumulation, and hyperinnervation after ten challenges. RESULTS: Oxazolone-challenged mice developed significant tactile sensitivity that persisted for over three weeks after labiar allergen exposures ceased. Allergic sites were characterized by mast cell accumulation, sensory hyper-innervation and infiltration of regulatory CD4+CD25+FoxP3+ T cells as well as localized early increases in transcripts encoding Nerve Growth Factor and nerve-mast cell synapse marker Cell Adhesion Molecule 1. Local depletion of mast cells by intra-labiar administration of secretagogue compound 48/80 led to a reduction in both nerve density and tactile sensitivity. CONCLUSIONS: Mast cells regulate allergy-provoked persistent sensitivity to touch. Mast cell-targeted therapeutic strategies may provide novel means to manage and limit chronic pain conditions associated with atopic disease.

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

Adoptive transfer of mast cells does not enhance the impaired survival of Kit(W)/Kit(W-v) mice in a model of low dose intraperitoneal infection with bioluminescent Salmonella typhimurium.

Mast cells are important effector cells in IgE-associated immune responses, but also can contribute to host defense in certain examples of bacterial infection. We found that genetically mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice exhibited more bacterial CFUs per spleen by 6 days after intraperitoneal injection of bioluminescent Salmonella typhimurium, and died more rapidly after infection, than did the congenic WBB6F1-Kit(+/+) wild type mice. Adoptive transfer of bone marrow-derived cultured mast cells of Kit(+/+) origin to the peritoneal cavity of Kit(W)/Kit(W-v) mice resulted in engraftment of mast cells in the peritoneal cavity and mesentery of the recipient mice, and the development of large numbers of mast cells in the spleen. However, such mast cell-engrafted Kit(W)/Kit(W-v) mice appeared sicker after intraperitoneal injection with S. typhimurium than did mast cell-deficient Kit(W)/Kit(W-v) mice, and exhibited numbers of CFUs of bacteria per spleen, and a survival curve, that were not significantly different than those of Kit(W)/Kit(W-v) mice. These results, when taken together with prior studies investigating the roles of mast cells in innate immunity, strongly suggest that whether mast cells can be shown to have a significant role in enhancing survival during bacterial infections may depend critically on the details of the particular experimental systems examined.

Mast cells: versatile gatekeepers of pain.

Mast cells are important first responders in protective pain responses that provoke withdrawal from intense, noxious environmental stimuli, in part because of their sentinel location in tissue-environment interfaces. In chronic pain disorders, the proximity of mast cells to nerves potentiates critical molecular cross-talk between these two cell types that results in their synergistic contribution to the initiation and propagation of long-term changes in pain responses via intricate signal networks of neurotransmitters, cytokines and adhesion molecules. Both in rodent models of inflammatory pain and chronic pain disorders, as well as in increasing evidence from the clinic, it is abundantly clear that understanding the mast cell-mediated mechanisms underlying protective and maladaptive pain cascades will lead to improved understanding of mast cell biology as well as the development of novel, targeted therapies for the treatment and management of debilitating pain conditions.

Clonal differences in IgE antibodies affect cutaneous anaphylaxis-associated thermal sensitivity in mice.

Cellular and molecular mediators of immune responses are increasingly implicated in acute and chronic pain pathophysiologies. Here we demonstrate that passive cutaneous IgE/Ag anaphylaxis provokes increased thermal sensitivity in the hind paw tissue of mice. The murine anti-DNP IgE antibodies SPE-7 and ɛ26 are known to induce differential cytokine production in bone marrow cultured mast cells in vitro without antigen challenge. We found a novel, antigen-dependent heterogeneity in the thermal pain responses elicited in the hind paws between SPE-7 and ɛ26 sensitized DNP-challenged mice. Mice experienced pronounced hind paw thermal sensitivity lasting 6h after DNP challenge when sensitized with SPE-7 but not ɛ26 IgE. The two IgE clones induced equivalent hind paw edema, neutrophil influx, cytokine production, and reduction in tissue histamine content in vivo, and bound to the same or overlapping epitopes on the DNP antigen in vitro. Therefore IgE antibodies against the same antigen can induce comparable inflammation, yet contribute to markedly different anaphylaxis-associated pain within an allergic response, suggesting that non-canonical IgE binding partners such as sensory neurons may play a role in allergy-related pain responses.

Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen, or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.