Characteristic findings of cervical Papanicolaou tests from transgender patients on androgen therapy: Challenges in detecting dysplasia.

INTRODUCTION: The characteristic features of Papanicolaou (Pap) tests collected from female-to-male (FTM) transgender patients on androgen therapy have not been well defined in the literature. FTM transgender patients require cervical cancer screening with the same recommended frequency as cis-gender females. Dysplasia remains challenging to differentiate from atrophy. Without pertinent history, the atrophic findings in younger transgender patients can be misinterpreted as high-grade dysplasia. METHODS: A review of all cervical Pap tests of transgender patients receiving androgen therapy (2010-2017) was performed. Bethesda diagnosis, cytomorphological features, HPV testing and cervical biopsy results were reviewed. RESULTS: Eleven transgender patients receiving androgen therapy were identified with 23 cervical Pap tests, 11 HPV tests and five cervical biopsies performed. A review of the Pap tests demonstrated: 57% negative for intraepithelial lesion; 13% unsatisfactory; 13% atypical squamous cells of undetermined significance; 13% atypical squamous cells - cannot exclude high-grade squamous intraepithelial lesion; and 4% high-grade squamous intraepithelial lesion. The rates of abnormal tests were higher than our age-matched cis-gender atrophic cohort rates of unsatisfactory (0.5%), atypical squamous cells of undetermined significance (7%), atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (0%) and high-grade squamous intraepithelial lesion (0.5%). The cytological findings from liquid-based preparations included dispersed and clustered parabasal-type cells, scattered degenerated cells, smooth evenly dispersed chromatin, and occasional mild nuclear enlargement and irregularity. Dysplastic cells had larger nuclei, hyperchromatic clumped chromatin, and more irregular nuclear contours. CONCLUSIONS: The evaluation of dysplasia can be challenging on Pap tests from transgender patients on androgen therapy. The cohort evaluated had higher rates of unsatisfactory and abnormal Pap tests. Pathologists should be familiar with the distinctive cytomorphological changes in the Pap tests from patients on androgen therapy to evaluate them appropriately.

Thymic and extrathymic contributions to T helper cell function in murine neonates.

Murine neonatal CD4+ responses are often biased to Th2 function. There is increasing evidence that this phenomenon may be regulated both at the level of the thymus and the peripheral lymphoid compartment. In particular, residual fetal influence on the neonatal thymus may lead to an imprinting of developing T cells that is maintained in CD4+ cells when they emigrate to peripheral organs. Such imprinting may involve epigenetic modification of the Th2 cytokine gene locus and acquisition of the capacity to undergo rapid cell cycling. These properties, coupled with the homeostatic proliferation occurring in the peripheral tissues of neonates, shape a CD4+ population with the capacity for enhanced Th2 responsiveness.

Choroid plexus selectively accumulates T-lymphocytes in normal controls and after peripheral immune activation.

We determined T-lymphocyte migration into brain and choroid plexus (CPx) after enterotoxin-induced systemic immune activation. CPx T-lymphocytes/mm2 in control mice were > 3 logs more numerous than brain and increased by as much as 150-fold by post-enterotoxin Day 3 (p < 0.01). Flow cytometry of pooled CPx confirmed post-enterotoxin increases. Brain T-lymphocytes increased up to 17-fold after SEB and accumulated in subependymal and periventricular brain. T cell apoptosis was absent. These results show preferential T-lymphocyte migration to CPx over brain and suggest that brain T cells may be derived from the CPx by direct migration or by cerebrospinal fluid dissemination.

CD4+ and CD8+ cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis.

To test the hypothesis that CD4+ T lymphocytes accumulate in brains of end-stage acquired immunodeficiency syndrome (AIDS) patients, we examined T-lymphocyte subsets in the CA1, CA3, and CA4 regions of the hippocampus of AIDS patients with (n = 10) and without (n = 11) human immunodeficiency virus encephalitis (HIVE) plus controls (n = 7). HIV p24 antigen was common in monocytic cells and rare in activated/memory CD45RO+ lymphocytes. Hippocampal activated/memory CD45RO+ T lymphocytes significantly increased (P <.001) in seven of the eight hippocampal subregions with hippocampal HIVE (1.14 +/- 1.4 T cells/high-power field [hpf]), but AIDS hippocampus without HIVE were similar to controls (0.03 +/- 0.07 T cells/hpf and 0.03 +/- 0.09 T cells/hpf, respectively). CD45RO+ and CD3+ lymphocytes were similar in numbers and distribution, whereas CD4+ and CD8+ lymphocytes were weakly immunoreactive and less frequent. All four lymphocyte subtypes were present in perivascular spaces and microglial nodules of HIVE, and had direct contact with neurons. Monocytes, microglia, and multinucleated giant cells were immunoreactive for CD4 in AIDS cases with hippocampal HIVE but microglia in remaining AIDS cases and controls were CD4-. CD68+ macrophages significantly increased in hippocampus of HIVE patients (P <.05) and were predominately perivascular in the absence of local HIVE. These studies show that CD4+ T lymphocytes, as well as CD8+ T lymphocytes, participate in the local inflammatory response of HIVE in end-stage AIDS patients, and suggest that their recruitment requires local HIV infection. The perineuronal location of CD4+ cells provides the potential for lymphocyte-mediated neuronal injury or trans-receptor-mediated neuronal infection.

Toxin levels in serum correlate with the development of staphylococcal scalded skin syndrome in a murine model.

Staphylococcal scalded skin syndrome (SSSS) is an exfoliative dermatitis that results from infection with exfoliative toxin-producing Staphylococcus aureus. SSSS is seen primarily in infants and children. Here we ask if there is a specific maturation process that protects healthy adults from this syndrome. For these studies, an active recombinant exfoliative toxin A (rETA) was used in a neonatal mouse model. A time course generated on the susceptibility to the toxin as a function of mouse age indicated that BALB/c mice developed the characteristic symptoms of SSSS until day 7 of life. Between day 7 and day 8 of life there was a dramatic decrease in susceptibility, such that mice at day 9 of life were resistant to the effects of the toxin. This time course corresponds approximately to the time needed for maturation of the adaptive immune response, and SSSS in adults is often identified with immunocompromised states. Therefore, mice deficient in this response were examined. Adult mice thymectomized at birth and adult SCID mice did not develop the symptoms of SSSS after injection with the toxin, indicating that the adaptive immune response is not responsible for the lack of susceptibility observed in the older mice. SSSS in adults is also associated with renal disorders, suggesting that levels of toxin in serum are important in the development of the disease. rETA was not cleared as efficiently from the serum of 1-day-old mice compared to clearance from 10-day-old mice. Ten-day-old mice were given repeated injections of toxin so that the maximal level of toxin was maintained for a sustained period of time, and exfoliation occurred in these mice. Thus, whereas the adaptive immune response is not needed for protection of adult mice from SSSS, efficient clearance of the toxin from the bloodstream is a critical factor.

The generation of Th memory in neonates versus adults: prolonged primary Th2 effector function and impaired development of Th1 memory effector function in murine neonates.

Immunization during the neonatal period often results in Th2-biased secondary responses. To understand the regulation of this phenomenon, we have examined all phases of Th development, from the generation of primary effectors to the duration of the primary effector stage to the production of memory effector function. First, we had previously reported that although primary responses in the neonatal lymph nodes are mature, mixed Th1/Th2-like, primary responses in the spleens of the same animals are exclusively Th2-like. To determine whether Th2-dominant secondary responses are due to the Th2-polarized primary function in the spleen, neonates were splenectomized before immunization. Even in the absence of primary neonatal splenic responses, the secondary responses of neonates were Th2 dominant. Thus, the overwhelmingly Th2 primary responses in the neonatal spleen are not required to generate Th2-dominant memory in the lymph nodes. Second, we have compared the kinetics of the primary response phase in neonates and adults. In adults, Ag-specific Th2 function disappeared rapidly from both the lymph nodes and spleen. In contrast, primary Th2 function persisted out to 5 wk in both neonatal organs. Third, the generation of Th memory responses was examined in animals initially immunized as neonates and in adults. These experiments demonstrated that neonates are selectively impaired in the development of Th1 memory effector function. Together, these results indicate that neonates are biased to Th2 function at all phases of an immune response.

Early block in maturation is associated with thymic involution in mammary tumor-bearing mice.

We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.

Development of neonatal Th1/Th2 function.

Newborn animals generally mount poor T cell-mediated immune responses in vivo. As a result, neonates fall prey to infectious agents and diseases which have little impact on immunocompetent adult animals. For some time, it was believed that this phenomenon was due to an intrinsic inability of newborns to mount developmentally mature Th1 responses. Recent studies in mice have challenged that view; under certain conditions, adult-level Th1 function has been achieved in newborns. More often, however, neonates develop Th2-dominant responses. A major challenge in the field of developmental immunology is to understand why the 'default' response for neonates is Th2 function. Cell intrinsic as well as environmental influences may contribute to Th2 skewing in neonates.

Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates.

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.

Chronic systemic administration of tumor necrosis factor alpha and HIV gp120: effects on adult rodent brain and blood-brain barrier.

Since tumor necrosis factor alpha (TNF-alpha) and HIV gpl20 glycoprotein are both neurotoxic, the possibility that systemic sources of these two agents mediate AIDS-associated blood-brain barrier (BBB) breakdown and brain damage was tested in two murine models: (1) intramuscular implantation of a TNF-alpha-transfected tumor in nu/nu mice and (2) daily subcutaneous injections of HIV gpl20 in BALB/c mice. The BBB remained intact; brain damage was not found, and apoptotic cell numbers did not increase. These results show that normal adult brain and BBB is unaffected by exposure to TNF-alpha or HIV gpl20 and suggest that severity of brain disease is not directly affected by systemic levels of these compounds.

T-cell function in newborn mice and humans.

Neonates mount poor immune responses, and it has been assumed that neonatal T cells differ qualitatively from adult T cells. Here, Becky Adkins discusses this issue in the light of recent data indicating that T cells in neonates are developmentally mature in their capacity to mount protective Th1-type and cytotoxic T lymphocyte responses.

Apoptosis of murine neonatal T cells.

Recent evidence supports the idea that T cells in neonatal animals are developmentally mature in their capacity to mount protective helper and cytotoxic responses. Nonetheless, neonates fall prey to infections which have little effect on adults and they often fail to mount mature responses to environmental, experimental, or vaccine antigens. To reconcile these observations, it may be important to consider the potential role of apoptosis in neonatal immune responses. In adults, apoptosis is well established as a centrally important process in the homeostasis of cellular immune responses. Activated T cells deprived of IL-2 undergo cytokine withdrawal-induced apoptosis. Previously activated T cells can also be triggered by secondary stimulation to undergo activation induced apoptosis. This review summarizes our current state of knowledge of apoptosis of murine neonatal T cells and discusses the possible impact(s) of this apoptosis on neonatal immune responses in vivo.

Autoantibodies to T-lineage cells in aged mice.

Aging is accompanied by a marked decline in protective immune function. This loss of effective immunity is largely due to alterations in the T-cell compartment. There are major impairments in both the production of new T-cells within the thymus and in the functions of mature T-cells in peripheral lymphoid organs. The mechanism(s) underlying this age-related decline in T-lineage cells is not clear. Here, we demonstrate that aging is accompanied by the appearance of appreciable titers of anti-T-lineage autoantibodies. The autoantibodies, which are exclusively of the IgM class, begin to appear at 1 year of life and are universally found in the sera of 2-year-old mice. Among thymocytes, all CD4/CD8 subsets reacted with the autoantibodies, with the CD4+8+ subset showing the greatest reactivity. The autoantibodies also bound to resting peripheral CD4+ and CD8+ cells. Following activation with either anti-CD3 or with TCR-independent stimulators, reactivity to peripheral T-cells was diminished, suggesting that the determinants recognized by the autoantibodies are downregulated in response to activation signals. Lastly, thymocytes freshly isolated from old, but not young, mice have IgM antibodies bound to their surfaces. Thus, circulating autoantibodies in old mice have access to the thymus and bind to thymocytes in situ. These results lead to the proposal that the presence of anti-T-lineage autoantibodies in vivo interferes with normal T-cell development and/or function in aged animals.

Monoclonal antibodies to the common gamma-chain as cytokine receptor antagonists in vivo: effect on intrathymic and intestinal intraepithelial T lymphocyte development.

Mice lacking a functional gamma c subunit of cytokine receptors exhibit profound defects in the development of multiple lymphoid lineages. To investigate the role of gamma c-dependent cytokines in T cell development, the phenotype of developing T cells was compared in interleukin (IL)-7Ralpha-deficient mice and anti-gamma c mAb-treated chimeric mice reconstituted with adult bone marrow cells or subsets of pro-T cells. These studies indicate that gamma c contributes to T cell development at multiple stages of pro-T cell maturation and that IL-7/IL-7R is the primary cytokine for thymic-dependent T cell development. However, our data also implicate other gamma c-dependent cytokines during thymic T cell development. By contrast, substantial intestinal intraepithelial lymphocytes (IEL) development was observed in the intestinal intraepithelium in both types of mice. Analysis of IL-7Ralpha-deficient mice indicates that the IL-7/IL-7R system is critical only for the development of TCR gammadelta+ IEL. However, the inhibitory activity of the anti-deltac mAb in the chimeric mouse model suggests that additional gamma cutilizing cytokines regulate the development of the remaining subsets of IEL.

Newborn mice develop balanced Th1/Th2 primary effector responses in vivo but are biased to Th2 secondary responses.

Newborn mice are impaired in their abilities to mount protective immune responses. For decades, it was generally held that the poor responses of newborns were largely due to the developmentally immature state of the T cells. In vitro studies showing that neonatal T cells were deficient in Th1 cytokine production, proliferation, and secondary responsiveness strongly supported this idea. Recently, several studies have challenged this view; animals exposed to Ag as neonates were shown to have mature Th1 responses in adulthood. However, it is not clear whether the mature immune responses were actually mounted by T cells generated after the neonatal stage. We have reexamined this issue by analyzing the capabilities of neonatal lymph node T cells to develop into Ag-specific effector cells during the actual neonatal period. Our results demonstrate that the capacity to develop a balanced Th1/Th2 primary effector response is fully mature within the first week of life. However, while neonatal and adult primary cytokine profiles were very similar, Th2 secondary responses predominated in animals first immunized as newborns. Moreover, we have observed other differences between adults and neonatal responses, including 1) the kinetics of cytokine production and responsiveness to adjuvant during the primary response, and 2) the contribution of spleen and lymph node to secondary responses. We propose that these differences reflect developmental regulation of effector cell function that has important consequences to neonatal immune function.

Perforated cecal adenocarcinoma presenting as a thigh abscess.

Complicated colorectal carcinoma has several symptoms, the most common being bleeding and obstruction. Occasionally it will cause perforation, which carries a worse prognosis. We report a case of perforated adenocarcinoma of the cecum that presented as an abscess of the thigh. We also present a review of the literature on this subject.

The common gamma-chain of cytokine receptors regulates intrathymic T cell development at multiple stages.

Signaling through the common gamma chain (gamma c), a subunit of the receptors for IL-2, -4, -7, -9, and -15, is critical for lymphocyte development, with the IL-7/IL-7R representing one important interaction. To investigate the stages of intrathymic T cell development that are dependent on gamma c and to determine whether gamma c controls T cell development solely as a component of the IL-7R, intrathymic T cell development was compared in IL-7R alpha-deficient mice and anti-gamma c-treated chimeric mice reconstituted with bone marrow and purified pro-T cells. In the presence of anti-gamma c, each of four phenotypically distinguishable stages of CD4- CD8- thymocytes failed to reconstitute T cell development, suggesting that each of these subsets of pro-T cells required gamma c for their differentiation and/or growth. Reconstitution of anti-gamma c-treated chimeric mice with bone marrow from IL-7R alpha-deficient mice indicated that IL-7R only partially contributed to intrathymic T cell development. Furthermore, when compared with IL-7R-deficient mice, anti-gamma c chimeric and gamma c-deficient mice exhibited a distinct phenotypic pattern of pro-T cell development. Collectively, these results indicate that several gamma c-sharing cytokines may contribute to T cell development in the thymus and suggest that one of these cytokines may be novel.

The tyrosine photophysics of a primase-derived peptide are sensitive to the peptide's zinc-bound state: proof that the bacterial primase hypothetical zinc finger sequence binds zinc.

A 35-amino acid peptide corresponding to the putative "zinc finger" sequence of primase was prepared to study its zinc binding properties. When zinc was added to the peptide, it was found that the fluorescence quantum yield of the single tyrosine increased by 46% and the average lifetime by 34%. The binding stoichiometry was one zinc per peptide. Below pH 6.0 and above pH 8.5, the zinc-peptide binding affinity was less than 1 microM and could be accurately determined. Interpolation from those binding constants suggested that the affinity at pH 7.5 was between 10 and 100 nM. The absorption spectrum of the cobalt(II)-peptide complex was consistent with tetrahedral metal coordination by three sulfur and one imidazole nitrogen ligands. The peptide affinity for cobalt was less than for zinc, indicating metal specificity. Analysis of the fluorescence intensity pH profile, circular dichroism spectra, the effect of extrinsic quenchers indicated that at neutral pH (1) the free peptide up into a structure to place the tyrosine in an environment protected from solvent, (2) the peptide bound zinc via its three cysteines and one of its histidines resulting in little change to the polypeptide secondary structure or to the tyrosine solvent accessibility, and (3) when the peptide bound zinc, it bound directly to or caused the immobilization of the groups that had been intramolecularly collisionally quenching the tyrosine which resulted in the observed increases in tyrosine quantum yield and lifetime.

PBPK modeling/Monte Carlo simulation of methylene chloride kinetic changes in mice in relation to age and acute, subchronic, and chronic inhalation exposure.

During a 2-year chronic inhalation study on methylene chloride (2000 or 0 ppm; 6 hr/day, 5 days/week), gas-uptake pharmacokinetic studies and tissue partition coefficient determinations were conducted on female B6C3F1, mice after 1 day, 1 month, 1 year, and 2 years of exposure. Using physiologically based pharmacokinetic (PBPK) modeling coupled with Monte Carlo simulation and bootstrap resampling for data analyses, a significant induction in the mixed function oxidase (MFO) rate constant (Vmaxc) was observed at the 1-day and 1-month exposure points when compared to concurrent control mice while decreases in glutathione S-transferase (GST) rate constant (Kfc) were observed in the 1-day and 1-month exposed mice. Within exposure groups, the apparent Vmaxc maintained significant increases in the 1-month and 2-year control groups. Although the same initial increase exists in the exposed group, the 2-year Vmaxc is significantly smaller than the 1-month group (p < 0.001). Within group differences in median Kfc values show a significant decrease in both 1-month and 2-year groups among control and exposed mice (p < 0.001). Although no changes in methylene chloride solubility as a result of prior exposure were observed in blood, muscle, liver, or lung, a marginal decrease in the fat:air partition coefficient was found in the exposed mice at p = 0.053. Age related solubility differences were found in muscle:air, liver:air, lung:air, and fat:air partition coefficients at p < 0.001, while the solubility of methylene chloride in blood was not affected by age (p = 0.461). As a result of this study, we conclude that age and prior exposure to methylene chloride can produce notable changes in disposition and metabolism and may represent important factors in the interpretation for toxicologic data and its application to risk assessment.

Naive murine neonatal T cells undergo apoptosis in response to primary stimulation.

The cellular mechanisms controlling deficient immune responses in newborn animals are not well understood. Our earlier studies showed that developmental regulation of Th cell activity may be one of the major causes of immunodeficiency in neonatal animals. Naive murine neonatal T cells showed poor Th1 activity but robust Th2 activity in response to primary stimulation in vitro. Although they produced high levels of IL-4, neonatal T cells proliferated poorly, suggesting that neonatal T cell survival in primary cultures may be limited. We show here that, unlike adult T cells, naive neonatal T cells undergo apoptosis in response to primary TCR-mediated stimulation. Ligation of the TCR alone, in the absence of accessory cell costimulation, is sufficient to induce apoptosis. Moreover, CD4+ and CD8+ T cells show equivalent levels of apoptosis. Lastly, this apoptosis can be prevented by the addition of excess IL-2 or by conditions promoting a high level of IL-2 production (TCR-independent stimulation, anti-CD28 mAb, or exogenous IL-6) by neonatal T cells. However, IL-2 alone is not sufficient to support functional rescue from apoptosis; only IL-6 supports the ability of these cells both to survive and to mount vigorous secondary responses. The identification of conditions allowing functional rescue from apoptosis in vitro has important implications for enhancing vaccine responsiveness in vivo.