Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats.

Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.

Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents.

Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 µM) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MTT assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.

Knockout of histidine decarboxylase decreases bile duct ligation-induced biliary hyperplasia via downregulation of the histidine decarboxylase/VEGF axis through PKA-ERK1/2 signaling.

Histidine is converted to histamine by histidine decarboxylase (HDC). We have shown that cholangiocytes 1) express HDC, 2) secrete histamine, and 3) proliferate after histamine treatment via ERK1/2 signaling. In bile duct-ligated (BDL) rodents, there is enhanced biliary hyperplasia, HDC expression, and histamine secretion. This studied aimed to demonstrate that knockdown of HDC inhibits biliary proliferation via downregulation of PKA/ERK1/2 signaling. HDC(-/-) mice and matching wild-type (WT) were subjected to sham or BDL. After 1 wk, serum, liver blocks, and cholangiocytes were collected. Immunohistochemistry was performed for 1) hematoxylin and eosin, 2) intrahepatic bile duct mass (IBDM) by cytokeratin-19, and 3) HDC biliary expression. We measured serum and cholangiocyte histamine levels by enzyme immunoassay. In total liver or cholangiocytes, we studied: 1) HDC and VEGF/HIF-1α expression and 2) PCNA and PKA/ERK1/2 protein expression. In vitro, cholangiocytes were stably transfected with shRNA-HDC plasmids (or control). After transfection we evaluated pPKA, pERK1/2, and cholangiocyte proliferation by immunoblots and MTT assay. In BDL HDC(-/-) mice, there was decreased IBDM, PCNA, VEGF, and HDC expression compared with BDL WT mice. Histamine levels were decreased in BDL HDC(-/-). BDL HDC(-/-) livers were void of necrosis and inflammation compared with BDL WT. PKA/ERK1/2 protein expression (increased in WT BDL) was lower in BDL HDC(-/-) cholangiocytes. In vitro, knockdown of HDC decreased proliferation and protein expression of PKA/ERK1/2 compared with control. In conclusion, loss of HDC decreases BDL-induced biliary mass and VEGF/HIF-1α expression via PKA/ERK1/2 signaling. Our data suggest that HDC is a key regulator of biliary proliferation.

Inhibition of histidine decarboxylase ablates the autocrine tumorigenic effects of histamine in human cholangiocarcinoma.

BACKGROUND: In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1-H4 histamine receptors (HRs). OBJECTIVE: To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth. METHODS: In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment. RESULTS: Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1-H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (~80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups. CONCLUSION: The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.

Progression from atypical/dysplastic intraepidermal proliferations and carcinoma in situ to invasive tumors: a pathway based on current knowledge.

Oncology research efforts in recent years have begun to elucidate the role of the peritumoral stroma in the development of dysplasia and subsequent invasive malignancy. In the skin, the stroma surrounding keratinocytic and melanocytic tumors reacts to the dysplastic epidermis in a similar fashion to the wound healing response. Once epidermal genetic mutations and aberrant molecular signaling have occurred, the stroma responds through a 3-phase process-extracellular matrix degradation is produced by matrix metalloproteinases; angiogenesis is induced by vascular endothelial growth factor and mast cell mediators; and the inflammatory response is elicited by cytokines and cyclin D1 overexpression balanced by the immunosuppression of mast cell mediators such as tumor necrosis factor alpha, histamine, and transforming growth factor beta. By reacting like injured dermis, the actions of various stromal mediators directly allow for, and even encourage, the progression of in situ atypia/dysplasia to invasive malignancy. The intent of this article is to review the multistep biological and chemical stromal processes, which are involved in the progression of atypical/dysplastic intraepidermal proliferations to invasive malignancy.

Nodular mucinosis of the breast: expanding our understanding with an unusual case.

Nodular mucinosis of the breast is an extraordinarily rare lesion that occurs in patients who do not have Carney syndrome. Typically, the affected patients are young women with no medical history and a nodule under 1 nipple. Histopathologic examination has, until now, shown a multinodular myxoid lesion containing scattered capillaries and histiocytes but void of epithelial components. We present the case of a 72-year-old woman with a history of mucinous carcinoma of the breast who now presents with a painful subareolar nodule of the same breast. Biopsy and histological examination confirmed nodular mucinosis of the breast.

Successful pregnancy in a hemodialysis patient and marked resolution of her nephrogenic systemic fibrosis.

Nephrogenic systemic fibrosis (NSF) is a recently recognized clinicopathologic entity. It is believed to be related to exposure to gadolinium-containing magnetic resonance imaging agents with gadolinium deposition in the tissues, including skin and other organs. It mainly affects patients on dialysis therapy. Pregnancy in dialysis patients is a rare occurrence. We present a case of a dialysis patient who developed NSF after exposure to gadodiamide and went on to have a successful pregnancy while on hemodialysis therapy. The patient had marked clinical and histological improvement in NSF during and after her pregnancy. This also correlated with decreasing gadolinium levels in skin biopsy tissue specimens. We discuss the interplay of factors involved in the successful pregnancy and improvement in NSF lesions in this patient.

Quantification of gadolinium in nephrogenic systemic fibrosis: re-examination of a reported cohort with analysis of clinical factors.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) has been associated with exposure to gadolinium-based contrast agent (GdCA) used in medical imaging. OBJECTIVE: We sought to quantify levels of gadolinium in affected skin of patients with NSF and correlate the levels to clinical and laboratory parameters. METHODS: Skin biopsy specimens were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS) and the micrograms of gadolinium per gram (microg/g) of dry tissue were determined. Clinical and laboratory data were obtained through retrospective chart review. Pearson correlation coefficients were used to correlate tissue gadolinium levels with various parameters. RESULTS: Six patients from a prior cohort were analyzed for gadolinium in affected skin. The mean amount of gadolinium in affected skin of NSF patients was 320.1 microg/g. No gadolinium was found in limited control tissue from patients unexposed to GdCA or in patients exposed to such agents, but without disease. Higher levels of gadolinium in skin correlated with younger age, lower body weight, lower corrected serum calcium levels, and lower erythropoietin dosing. LIMITATIONS: The study was limited by the small number of cases and by the retrospective nature of the data and skin samples. CONCLUSION: Gadolinium is present in substantial amounts within the skin of patients with NSF. Quantification of gadolinium in tissue using ICP-MS may facilitate our understanding of the disease.

Prevalence of the human papillomavirus in an inner-city indigent population with previously normal Pap tests.

OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) in our inner-city indigent population (clinic population) of women with previously normal Pap tests and to identify any associated risk factors. MATERIALS AND METHODS: A prospective cohort of 187 women between the ages of 15 and 49 years, with previously normal Pap tests, was recruited from a university affiliated outpatient clinic. A demographic questionnaire of social and sexual history was elicited, and ThinPrep cytology (Cytyc, Marlborough, MA) and HPV Digene Hybrid Capture II results (Digine, Gaithersburg, MD) were obtained. RESULTS: The prevalence of HPV in our primarily Hispanic clinic population was 21%. The mean age of women with HPV was 28.9 years and those without were 32.1 years (p <.046). In women with HPV, 24% had abnormal Pap tests, whereas in those without HPV, 5% had abnormal Pap tests (p <.001). Women who were older and parous were less likely to have HPV (7.5%; p <.024). The presence of HPV was not influenced by sexual behaviors, sexually transmitted diseases, smoking, race, or contraceptive use. CONCLUSIONS: The prevalence of HPV in an inner-city indigent population, despite previously normal cytology, was consistent with earlier reported rates of HPV. Our data suggest that younger, nulliparous women have a high prevalence of HPV.

Nephrogenic systemic fibrosis: a review of 6 cases temporally related to gadodiamide injection (omniscan).

OBJECTIVES: Nephrogenic systemic fibrosis (NSF) is a rare acquired disorder that was first recognized in 1997. Presented is a retrospective review of 6 cases of NSF diagnosed by skin biopsy in our institution during the past 4 years and their relationship to gadodiamide exposure. METHODS AND RESULTS: Patient age ranged from 23 to 71 years. The onset of symptoms consistent with NSF was between 19 days and 2 months after gadodiamide exposure. Five patients had severe renal impairment and started dialysis around the period of gadodiamide exposure (1 day before the 37 days after contrast administration). The sixth patient had a clotted access at the time of a contrast-enhanced magnetic resonance venogram and was hence not being adequately dialyzed. The dose of gadodiamide ranged from 16 to 40 mL (0.11 to 0.36 mmol/kg). Despite having normal serum bicarbonate, 5 of the 6 patients had an elevated anion gap metabolic acidosis. CONCLUSIONS: In our 6 patients, all had either failing native or transplant kidneys at the time of gadodiamide exposure. The development of NSF was temporally related to gadodiamide injection, suggesting as the etiology dechelation of the agent and thus emphasizing the need for change in clinical practice.

Dysplasia associated with atypical glandular cells on cervical cytology.

OBJECTIVES: To estimate the rates of and identify risk factors for disease in women with atypical glandular cells of undetermined significance (AGUS). METHODS: From 1998-2001, 477 Pap tests at Hartford Hospital were classified as AGUS and met the inclusion criteria of this study. Findings were evaluated for 2 years from the initial test. Disease was defined as histology results with a finding of high-grade squamous intraepithelial lesion or greater. RESULTS: Disease was diagnosed in 9% of the women, including malignancy in 3%. Women with malignant-appearing AGUS Pap tests had a higher rate of disease (29%) than women with benign-appearing (5%, P < .01) and unspecified AGUS Pap tests (13%, P < .03). Malignancies were associated with all subclassifications of AGUS Pap tests. Women aged less than 35 years were more likely to have disease than women aged 35 years or older (P < .02). Most women aged younger than 35 years had a squamous abnormality, whereas women aged 35 years or older had a greater diversity of squamous and glandular lesions and accounted for all cases of endometrial cancer, adenocarcinoma in situ, and cervical adenocarcinoma. Women with persistent AGUS Pap tests had a 31% rate of disease. The rate of disease among women with AGUS Pap tests collected by liquid-based cytology was 11%, compared with 6% among samples collected by the conventional method. CONCLUSION: These data suggest that women with atypical glandular cells are at substantial risk for dysplasia and malignancy. The rate of disease varies with the method of Pap test collection, age, presence of persistent AGUS Pap tests, and AGUS subclassification.

Lack of adherence to practice guidelines for women with atypical glandular cells on cervical cytology.

OBJECTIVE: We sought to estimate the rates and types of evaluation in women with atypical glandular cells of undetermined significance (AGC-US) on cervical cytology and to assess these findings on the basis of published management guidelines. METHODS: The rates of histologic sampling, comprehensive initial evaluations, and secondary evaluations were assessed in 477 women with an AGC-US Pap test from 1998 to 2001. A comprehensive evaluation was defined as a colposcopy and an endocervical curettage with or without a cervical biopsy. For women aged 35 or older, a comprehensive evaluation also included an endometrial biopsy. A secondary evaluation consisted of a diagnostic cone biopsy. RESULTS: Sixty-four percent of women with an AGC-US Pap test had histologic sampling; 36% were followed by repeat Pap test only. Thirty-six percent of women with an AGC-US Pap test had a comprehensive evaluation. Women with an AGC-US Pap test that was subclassified as malignant-appearing had higher rates of histologic and comprehensive evaluations than women with a benign-appearing or unspecified AGC-US Pap test (P < .01). Twenty-eight percent of women aged 35 or older had comprehensive evaluations compared with 57% of women younger than the age of 35 (P < .01). Secondary evaluations were performed in 8% of women with persistent AGC-US Pap tests and 2% of women with malignant-appearing AGC-US Pap tests after negative initial histologic evaluations. Twelve of the 42 cases of disease (29%) were diagnosed more than 1 year from the initial AGC-US Pap test. CONCLUSION: On the basis of accepted management guidelines, these data suggest that women with AGC-US Pap tests are undermanaged in both their initial and secondary evaluations.

Effect of a Decision Aid on Decision Making for the Treatment of Pelvic Organ Prolapse.

OBJECTIVES: The aim of this study was to evaluate if the addition of a decision aid (DA) decreases decisional conflict in women presenting for the management and treatment of pelvic organ prolapse (POP). METHODS: Women scheduled for the evaluation and management of POP were randomized into either of 2 groups: standard counseling (SC) alone (n = 51) or SC plus a DA (n = 53). Upon completion of their initial visit, patients filled out a 16-item decisional conflict scale and short form general health survey. Values were assessed for normality and compared between groups. Normally distributed, continuous data were evaluated with a Student t test. A χ2 test was used to compare selected categorical characteristics between groups. Differences in distributions of low and high decisional conflict were assessed with a Mann-Whitney U test. RESULTS: One hundred four women were randomized for this analysis. Baseline characteristics, including pelvic prolapse examination measurements, did not significantly differ between groups. The addition of a DA to SC did not significantly lower the level decisional conflict patients faced when deciding on a treatment plan (P = 0.566). There were no significant differences between groups in the following subscores: uncertainty, values clarity, support, effective decision, and informed. In addition, there were no between-group differences in choice of treatment plan (conservative management, pelvic floor physical therapy, pessary, and surgery; P = 0.835). CONCLUSIONS: In this relatively small sample, the addition of a DA to SC for women with POP does not significantly decrease the level of decisional conflict in making treatment-related decisions.

Toxicity and efficacy of benzamide riboside in cancer chemotherapy models.

Benzamide riboside (BR), a synthetic C-nucleoside, acts as a strong growth inhibitor of cancer cells in vitro and in vivo. BR, like TR and related nucleoside prodrugs, act by anabolism to NAD analogs. These analogs selectively inhibit IMPDH, leading to depletion of cellular GTP, growth cessation, and cell differentiation. To date only preclinical studies have been carried out. However, in tiazofurin (TR), a related drug, phase I/II clinical trials have been conducted in patients with acute leukemia and shown to be a very promising agent with a response rate of 85% in 26 patients in one of the trials. Tiazofurin is now undergoing phase III clinical trials as a result. Dose limiting toxicity of tiazofurin was headache, somnolence and nausea with no myelosuppression noted. By contrast, BR showed skeletal muscle toxicity, hepatotoxicity and myelosuppression in preclinical data. Skeletal muscle toxicity was noted in the paraspinal muscles and may represent dose-limiting toxicity. Since BR does exhibit myelosuppression, the most common chemotherapy-related side effect in humans, careful judgment is warranted should BR be included in multidrug regimens, although BR's potent cytotoxicity to tumor cells in preclinical models still makes it a promising drug.

Building an award-winning women's health ambulatory service and beyond.

Many barriers exist for the provision of high-quality health care to inner-city minority women. The barriers include access to care, compliance problems, financial concerns, system navigation issues, as well as language barriers. This article describes the transition of the Women's Ambulatory Health Services at Hartford Hospital from a traditional clinic model to a culturally sensitive private practice model. The road to transition was paved by valuable input from staff as well as patients. The final product was a much more efficient, inviting model that catered to the needs of the community.

Barriers to breastfeeding in a resident clinic.

Despite the known health benefits for mother and infant, compliance with exclusive breastfeeding continues to challenge many healthcare providers. In an ongoing attempt to maintain the goals of the Healthy People 2010 initiative, our institution set out to identify patients with suboptimal breastfeeding rates in order to recognize potential barriers. Review of breastfeeding rates at the time of discharge noted significantly lower participation by clinic patients. In order to develop successful interventions, the aim of this study was to survey clinic patients to determine their intentions, attitudes, and obstacles to the practice of exclusive breastfeeding. In total, 188 surveys were completed during a 2-month time period. Respondents were primarily Hispanic (76.4% vs. 9.6% black and 8.4% white) and multiparous (57.5%) with a mean age of 25.7 years (range, 15-39 years old). Although 95.3% of respondents indicated that they believed breastmilk provided adequate nutrition, only 35.3% planned on exclusively breastfeeding. Access to free formula through the Special Supplemental Nutrition Program for Women, Infants and Children was the most common reason not to breastfeed (48.3%), followed by fear of pain and the need to return to work/school. Patients reported that the person with the greatest influence on their decision to breastfeed was their partner/spouse. Access to a lactation counselor was the most popular intervention requested, even among experienced multiparous patients (78.9% of whom had previously breastfed). In conclusion, the survey indicated that planned exclusive breastfeeding rates are low among this inner-city resident clinic and interventions should include involvement of the partners/spouses and access to lactational support.

Successful surgical treatment of advanced erythema elevatum diutinum.

Erythema elevatum diutinum is a rare, chronic cutaneous vasculitis that presents with plaques or nodules on the extensor surfaces of extremities. Although the exact pathogenesis is unknown, patients usually have an underlying systemic medical problem such as malignancy, autoimmune disease or HIV. Management of the cutaneous manifestations is aimed at controlling the underlying disease process, in addition to medical therapy directed at suppressing the lesions. The difficult case of a 60-year-old man, who was not a candidate for medical therapy but has undergone successful surgical therapy of this rare disease for 10 years, is presented.

SERPINE 1 Links Obesity and Diabetes: A Pilot Study.

In the past decade there has been a dramatic increase in the number of Americans considered obese. Over this same period, the number of individuals diagnosed with diabetes has increased by over 40%. Interestingly, in a great number of cases individuals considered obese develop diabetes later on. Although a link between obesity and diabetes has been suggested, conclusive scientific evidence is thus far just beginning to emerge. The present pilot study is designed to identify a possible link between obesity and diabetes. The plasma proteome is a desirable biological sample due to their accessibility and representative complexity due, in part, to the wide dynamic range of protein concentrations, which lead to the discovery of new protein markers. Here we present the results for the specific depletion of 14 high-abundant proteins from the plasma samples of obese and diabetic patients. Comparative proteomic profiling of plasma from individuals with either diabetes or obesity and individuals with both obesity and diabetes revealed SERPINE 1 as a possible candidate protein of interest, which might be a link between obesity and diabetes.

Glatiramer acetate-associated, CD30+, primary, cutaneous, anaplastic large-cell lymphoma.

OBJECTIVE: To report the association of the development of a primary, cutaneous, anaplastic large-cell lymphoma after initiation of glatiramer acetate treatment of a patient with relapsing-remitting multiple sclerosis. DESIGN: Case report. SETTING: Dermatology outpatient clinic. Patient A 33-year-old white woman developed an erythematous nodule on her leg 4 months after starting treatment with glatiramer acetate. Biopsy showed primary, cutaneous, anaplastic large-cell lymphoma. Further evaluation showed no systemic involvement. Intervention Radiation therapy induced a complete remission. CONCLUSIONS: Several T-cell-mediated skin conditions have been associated with the use of glatiramer acetate, such as pseudolymphoma, drug eruptions, and erythema nodosum. We report the association of a T-cell malignancy with the use of glatiramer acetate.