A systematic review of the role of penicillin versus penicillin plus metronidazole in the management of peritonsillar abscess.

BACKGROUND: Peritonsillar abscess is a localised infection in the peritonsillar space. Pus from the abscess can contain anaerobes. Many clinicians prescribe metronidazole in addition to penicillin, but evidence to support this is limited. This review assessed the evidence of benefit of metronidazole for the treatment of peritonsillar abscess. METHODS: A systematic review was conducted of the literature and databases including Ovid Medline, Ovid Embase, PubMed and Cochrane library. Search terms included all variations of peritonsillar abscess, penicillin and metronidazole. RESULTS: Three randomised, control trials were included. All studies assessed the clinical outcomes after treatment for peritonsillar abscess, including recurrence rate, length of hospital stay and symptom improvement. There was no evidence to suggest additional benefit with metronidazole, with studies suggesting increased side effects. CONCLUSION: Evidence does not support the addition of metronidazole in first-line management of peritonsillar abscess. Further trials to establish optimum dose and duration schedules of oral phenoxymethylpenicillin would benefit clinical practice.

Does infection play a role in post-tonsillectomy haemorrhage? A narrative review.

OBJECTIVE: Post-tonsillectomy haemorrhage is an increasingly common cause of morbidity following tonsillectomy. Secondary post-tonsillectomy haemorrhage occurring more than 24 hours after an operation has long been attributed to post-operative infection; however, there is little evidence to support this hypothesis and the associated use of antibiotics in the current literature. METHOD: This study looked at the aetiology and evidence-based management of post-tonsillectomy haemorrhage, and investigated the impact of bacterial infection and antimicrobials on the pathogenesis and clinical course of this complication. RESULTS: A number of peri-operative risk factors for post-tonsillectomy haemorrhage exist, and infective pathologies, including recurrent or chronic tonsillitis and group A streptococcus on blood cultures, may predispose to bleeding. Very few studies have shown a link between post-tonsillectomy haemorrhage and objective markers of infection such as pyrexia, raised inflammatory markers or positive microbiology cultures. The role of antibiotics in secondary post-tonsillectomy haemorrhage remains controversial, and numerous randomised, controlled trials of peri-operative antibiotics have shown no significant difference in bleeding rates between antibiotics and controls. CONCLUSION: Further trials investigating the role of antibiotics and more robust studies investigating the presence of bacterial infection at the time of bleeding may be required to determine the true role of infection in post-tonsillectomy haemorrhage.

Diffusion-weighted magnetic resonance imaging for diagnosis of post-operative paediatric cholesteatoma.

OBJECTIVES: High rates of recidivism are reported after paediatric cholesteatoma surgery. Our practice has adapted to include non-echoplanar diffusion-weighted magnetic resonance imaging for the diagnosis of residual or recurrent cholesteatoma. This audit aimed to evaluate the performance of non-echoplanar diffusion-weighted magnetic resonance imaging in our paediatric population. METHODS: A retrospective review was conducted of non-echoplanar diffusion-weighted magnetic resonance imaging scans performed to detect residual disease or recurrence after surgery for cholesteatoma in children from 1 January 2012 to 30 November 2017 in our centre. Follow-up diffusion-weighted magnetic resonance imaging scans were reviewed to 16 August 2019. RESULTS: Thirty-four diffusion-weighted magnetic resonance imaging scans were included. The sensitivity and specificity values of diffusion-weighted magnetic resonance imaging for detecting post-operative cholesteatoma were 81 per cent and 72 per cent, respectively. Positive predictive and negative predictive values were 72 per cent and 81 per cent, respectively. CONCLUSION: Use of diffusion-weighted magnetic resonance imaging is recommended as a replacement for routine second-look surgical procedures in the paediatric population. However, we would caution that patients require close follow up after negative diffusion-weighted magnetic resonance imaging findings.

The impact of the COVID-19 pandemic on healthcare access and utilisation in South Sudan: a cross-sectional mixed methods study.

BACKGROUND: Indirect effects of the COVID-19 pandemic on communities in fragile and conflict-affected settings may be severe due to reduced access and use of healthcare, as happened during the 2015 Ebola Virus Disease outbreak. Achieving a balance between short-term emergency response and addressing long-term health needs is particularly challenging in fragile and conflict-affected settings such as South Sudan, given the already significant barriers to accessing healthcare for the population. This study sought to characterise the effect of COVID-19 on healthcare access and South Sudan's healthcare response. This can inform efforts to mitigate the potential impacts of COVID-19 or other epidemiological threats, and contribute to understanding how these may be balanced for greater health system resilience in fragile contexts. METHODS: We conducted a mixed methods study in three of South Sudan's states, combining data from a cross-sectional quantitative household survey with qualitative interviews and Focus Group Discussions. RESULTS: Even though some fears related to COVID-19 were reported, we found these did not greatly dissuade people from seeking care and do not yield significant consequences for health system programming in South Sudan. The pillars of the response focused on risk communication and community engagement were effective in reaching communities through different channels. Respondents and participants reported behaviour changes that were in line with public health advice. We also found that the implementation of COVID-19 response activities sometimes created frictions between the national government and international health actors, and that COVID-19 caused a greater reliance on, and increased responsibility for, international donors for health planning. CONCLUSIONS: Given the fact that global priorities on COVID-19 are greatly shifting, power dynamics between international health agencies and the national government may be useful to consider in further COVID-19 planning, particularly for the vaccine roll-out. South Sudan must now navigate a period of transition where COVID-19 vaccine roll-out continues and other domestic health burdens are re-prioritised.

Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

Planning considerations prior to laryngectomy for a patient infected with severe acute respiratory syndrome coronavirus-2 pre-operatively.

BACKGROUND: Coronavirus disease 2019 was declared a pandemic on 11th March 2020. All non-urgent surgical procedures have been postponed indefinitely. The British Association of Head and Neck Oncology state that only those with treatable head and neck cancer unsuitable for alternative treatment should undergo surgery. This paper details our management of a patient who tested positive for severe acute respiratory syndrome coronavirus-2 days before curative surgery for laryngeal cancer. CASE REPORT: By following British Association of Head and Neck Oncology guidance, a 49-year-old male scheduled for total laryngectomy and bilateral neck dissection for a T3 transglottic squamous cell cancer was pre-operatively identified as an asymptomatic carrier of severe acute respiratory syndrome coronavirus-2. Following 14-day isolation and laboratory proven viral clearance, he underwent successful major surgery. He was managed throughout the peri- and post-operative phases without complications or adverse effects on staff. CONCLUSION: With careful planning, previous coronavirus disease 2019 positive status should not prevent an individual from undergoing successful total laryngectomy and bilateral neck dissection in a safe and timely manner during the pandemic.

Severe acute respiratory syndrome coronavirus-2 in post-laryngectomy patients: case series of four patients.

OBJECTIVE: To report our experience of diagnosis, investigation and management in patients who had undergone laryngectomy secondary to previous squamous cell carcinoma, who were subsequently infected with severe acute respiratory syndrome coronavirus-2 during the coronavirus disease 2019 pandemic. CASE REPORTS: Four post-laryngectomy patients with laboratory-proven severe acute respiratory syndrome coronavirus-2 infection were admitted to our institution from 1 March to 1 May 2020. All patients displayed symptoms of coronavirus disease 2019 and underwent investigations, including swab and serum sampling, and chest X-ray where indicated. All were managed conservatively on dedicated coronavirus disease 2019 wards and were discharged without the requirement of higher level care. CONCLUSION: It is hypothesised that laryngectomy may offer a protective effect against severe or critical disease in severe acute respiratory syndrome coronavirus-2 infection. We hope sharing our experience will aid all practitioners in the management of this, often intimidating, cohort of patients.

Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome.

Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, α-tubulin, and ß-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 µg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in α-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes ß-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased ß-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells.

Wound protectors for improved exposure in open hernia repair.

INTRODUCTION: Achieving adequate surgical exposure is fundamental to good surgical practice. Traditionally, in the repair of ventral and open inguinal hernias, this has been accomplished with the aid of self-retaining retractors or by extending the incision length. We propose that using disposable wound protectors, surgical exposure will be improved for a given incision length in the repair of ventral and open inguinal hernias, compared to traditional methods. METHOD: Through the use of an animal tissue model, we compared incisions of varying lengths and measured the dimensions of the exposure achieved with disposable wound protectors (Alexis™) and a Mollison self-retainer. We calculated the surface area and, therefore, exposure gained using the specified wound retraction devices and compared the results. RESULTS: The average superficial surface area of wound with a self-retainer compared to the small and extra-small disposable wound protector was 2.65 cm2 vs 2.27 cm2 (small) and 2.2 cm2 (extra-small) for 2 cm incision length, 3.6 cm2 vs 4.93 cm2 and 4.2 cm2 for 3 cm incision length, 5.19 cm2 vs 8.25 cm2 and 6.27 cm2 for 4 cm incision length, 6.17 cm2 vs 12.25 cm2 and 9.07 cm2 for 5 cm incision length, and 8.75 cm2 vs 16.73 cm2 and 10.78 cm2 for 6 cm incision length [p = 0.038 (small) and p = 0.049 (extra-small)]. Our results show a statistically significant increase in surface area of a wound for each incision length when a disposable wound protector was used for exposure, compared to a self-retainer. CONCLUSION: Our results demonstrate that the use of disposable wound protectors provides superior surgical exposure for a given incision length compared to traditional techniques in the repair of ventral and open inguinal hernia repairs.

Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PURPOSE: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown. PATIENTS AND METHODS: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively). RESULTS: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031). CONCLUSION: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Histone Citrullination Represses MicroRNA Expression, Resulting in Increased Oncogene mRNAs in Somatolactotrope Cells.

Peptidylarginine deiminase (PAD) enzymes convert histone arginine residues into citrulline to modulate chromatin organization and gene expression. Although PADs are expressed in anterior pituitary gland cells, their functional role and expression in pituitary adenomas are unknown. To begin to address these issues, we first examined normal human pituitaries and pituitary adenomas and found that PAD2, PAD4, and citrullinated histones are highest in prolactinomas and somatoprolactinomas. In the somatoprolactinoma-derived GH3 cell line, PADs citrullinate histone H3, which is attenuated by a pan-PAD inhibitor. RNA sequencing and chromatin immunoprecipitation (ChIP) studies show that the expression of microRNAs (miRNAs) let-7c-2, 23b, and 29c is suppressed by histone citrullination. Our studies demonstrate that these miRNAs directly target the mRNA of the oncogenes encoding HMGA, insulin-like growth factor 1 (IGF-1), and N-MYC, which are highly implicated in human prolactinoma/somatoprolactinoma pathogenesis. Our results are the first to define a direct role for PAD-catalyzed histone citrullination in miRNA expression, which may underlie the etiology of prolactinoma and somatoprolactinoma tumors through regulation of oncogene expression.

Proton pump inhibitor and histamine-2 receptor antagonist use and risk of liver cancer in two population-based studies.

BACKGROUND: Proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H2RAs) are commonly used. PPIs have been shown to promote liver cancer in rats; however, only one study has examined the association in humans. AIMS: To investigate PPIs and H2RAs and risk of primary liver cancer in two large independent study populations. METHODS: We conducted a nested case-control study within the Primary Care Clinical Informatics Unit (PCCIU) database in which up to five controls were matched to cases with primary liver cancer, recorded by General Practitioners. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for associations with prescribed PPIs and H2RAs were calculated using conditional logistic regression. We also conducted a prospective cohort study within the UK Biobank using self-reported medication use and cancer-registry recorded primary liver cancer. Hazard ratios (HRs) and 95% CIs were calculated using Cox regression. RESULTS: In the PCCIU case-control analysis, 434 liver cancer cases were matched to 2103 controls. In the UK Biobank cohort, 182 of 475 768 participants developed liver cancer. In both, ever use of PPIs was associated with increased liver cancer risk (adjusted OR 1.80, 95% CI 1.34, 2.41 and adjusted HR 1.99, 95% CI 1.34, 2.94 respectively). There was little evidence of association with H2RA use (adjusted OR 1.21, 95% CI 0.84, 1.76 and adjusted HR 1.70, 95% CI 0.82, 3.53 respectively). CONCLUSIONS: We found some evidence that PPI use was associated with liver cancer. Whether this association is causal or reflects residual confounding or reverse causation requires additional research.

Microscopic colitis: a population-based case series over a 9-year period in Northern Ireland.

AIM: We report clinicopathological experience of microscopic colitis (MC) in a population-based case series in Northern Ireland over a 9-year period. METHOD: The pathology laboratory information system within a large teaching centre serving two healthcare trusts was interrogated for cases coded between 2008 and 2016 as collagenous colitis (CC) or lymphocytic colitis (LC). Demographic, clinical and follow-up information was collected from healthcare records. RESULTS: A total of 326 new diagnoses of MC were identified, an average annual incidence of 6.7 per 100 000 population. The average annual incidence of CC and LC was 5.0 and 1.7 per 100 000 population, respectively. For coding reasons it is likely that LC data are incomplete. Of 191 cases diagnosed by specialist gastrointestinal pathologists, 141 patients had CC and 50 patients had LC. Both CC and LC predominantly involved women aged 60-79. Some 15% demonstrated endoscopic abnormalities. Endoscopic sampling protocols varied widely: 30% of individuals with CC and 32% of those with LC had the right and left colon sampled separately, with histology concordant in 95% of cases. Of the 191 cases, only one case (of LC) was refractory to treatment; the rest exhibited a clinical response. Only 35 patients had follow-up endoscopy and biopsies, and three of each diagnosis showed persistent disease on histology. CONCLUSION: Overall, CC and LC are benign conditions with similar demographics, clinical associations, management and outcomes. Separate sampling of the right and left colon is advised at colonoscopy if this diagnosis is being considered, but left colonic sampling, which can be performed at flexible sigmoidoscopy, will diagnose the vast majority of cases.

A Model for Good Governance of Healthcare Technology Management in the Public Sector: Learning from Evidence-Informed Policy Development and Implementation in Benin.

Good governance (GG) is an important concept that has evolved as a set of normative principles for low- and middle-income countries (LMICs) to strengthen the functional capacity of their public bodies, and as a conditional prerequisite to receive donor funding. Although much is written on good governance, very little is known on how to implement it. This paper documents the process of developing a strategy to implement a GG model for Health Technology Management (HTM) in the public health sector, based on lessons learned from twenty years of experience in policy development and implementation in Benin. The model comprises six phases: (i) preparatory analysis, assessing the effects of previous policies and characterizing the HTM system; (ii) stakeholder identification and problem analysis, making explicit the perceptions of problems by a diverse range of actors, and assessing their ability to solve these problems; (iii) shared analysis and visioning, delineating the root causes of problems and hypothesizing solutions; (iv) development of policy instruments for pilot testing, based on quick-win solutions to understand the system's responses to change; (v) policy development and validation, translating the consensus solutions identified by stakeholders into a policy; and (vi) policy implementation and evaluation, implementing the policy through a cycle of planning, action, observation and reflection. The policy development process can be characterized as bottom-up, with a central focus on the participation of diverse stakeholders groups. Interactive and analytical tools of action research were used to integrate knowledge amongst actor groups, identify consensus solutions and develop the policy in a way that satisfies criteria of GG. This model could be useful for other LMICs where resources are constrained and the majority of healthcare technologies are imported.

Heller's myotomy and pneumatic dilatation in the treatment of achalasia: a population-based case-control study assessing long-term quality of life.

Long-term health-related quality-of-life (HRQL) outcomes have not been widely reported in the treatment of achalasia. The aims of this study were to examine long-term disease-specific and general HRQL in achalasia patients using a population-based case-control method, and to assess HRQL between treatment interventions. Manometrically diagnosed achalasia cases (n = 120) were identified and matched with controls (n = 115) using a population-based approach. Participants completed general (SF-12) and disease-specific (Achalasia Severity Questionnaire [ASQ]) HRQL questionnaires, as appropriate, in a structured interview. Mean composite scores for SF-12 (Mental Component Summary score [MCS-12] and Physical Component Summary score [PCS-12]) and ASQ were compared between cases and controls, or between intervention groups, using an independent t-test. Adjusted mean differences in HRQL scores were evaluated using a linear regression model. Achalasia cases were treated with a Heller's myotomy (n = 43), pneumatic dilatation (n = 44), or both modalities (n = 33). The median time from last treatment to HRQL assessment was 5.7 years (interquartile range 2.4-11.5). Comparing achalasia patients with controls, PCS-12 was significantly worse (40.9 vs. 44.2, P = 0.01), but MCS-12 was similar. However, both PCS-12 (39.9 vs. 44.2, P = 0.03) and MCS-12 (46.7 vs. 53.5, P = 0.004) were significantly impaired in those requiring dual treatment compared with controls. Overall however, there was no difference in adjusted HRQL between patients treated with Heller's myotomy, pneumatic dilatation or both treatment modalities. In summary, despite treatment achalasia patients have significantly worse long-term physical HRQL compared with population controls. No HRQL differences were observed between the treatment modalities to suggest a benefit of one treatment over another.

Nonlinear effects of potassium channel blockers on endothelium-dependent hyperpolarization.

In a number of published studies on endothelium-dependent hyperpolarization and relaxation, the results of the effects of K+ blockers have been difficult to interpret. When the effects of two blockers have been studied, often either blocker by itself had little effect, whereas the two blockers combined tended to abolish the responses. Explanations suggested in the literature include an unusual pharmacology of the K+ channels, and possible blocker binding interactions. In contrast, when we applied the same blockers to segments of small blood vessels under voltage clamp, the blockers reduced the endothelium-dependent K+ current in a linearly additive manner. Resolution of these contrasting results is important as endothelium-derived hyperpolarization (EDH) makes its greatest contribution to vasorelaxation in arterioles and small resistance arteries, where it can exert a significant role in tissue perfusion and blood pressure regulation. Furthermore, EDH is impaired in various diseases. Here, we consider why the voltage-clamp results differ from earlier free-running membrane potential and contractility studies. We fitted voltage-clamp-derived current-voltage relationships with mathematical functions and considered theoretically the effects of partial and total block of endothelium-derived K+ -currents on the membrane potential of small blood vessels. When the K+ -conductance was partially reduced, equivalent to applying a single blocker, the effect on EDH was small. Total block of the endothelium-dependent K+ conductance abolished the hyperpolarization, in agreement with various published studies. We conclude that nonlinear summation of the hyperpolarizing response evoked by endothelial stimulation can explain the variable effectiveness of individual K+ channel blockers on endothelium-dependent hyperpolarization and resulting relaxation.

Citrullination regulates the expression of insulin-like growth factor-binding protein 1 (IGFBP1) in ovine uterine luminal epithelial cells.

There are five peptidylarginine deiminase (PAD) isozymes designated as PADs 1, 2, 3, 4 and 6, and many are expressed in female reproductive tissues. These enzymes post-translationally convert positively charged arginine amino acids into neutral citrulline residues. Targets for PAD-catalyzed citrullination include arginine residues on histone tails, which results in chromatin decondensation and changes in gene expression. Some of the first studies examining PADs found that they are localized to rodent uterine epithelial cells. Despite these findings, the function of PAD-catalyzed citrullination in uterine epithelial cells is still unknown. To address this, we first examined PAD expression in uterine cross-sections from pregnant ewes on gestation day 25 (d25). Immunohistochemistry revealed that the levels of PADs 2 and 4 are robust in luminal and glandular epithelia compared with those of PADs 1 and 3. As PADs 2 and 4 have well-characterized roles in histone citrullination, we next hypothesized that PADs citrullinate histones in these uterine cells. Examination of caruncle lysates from pregnant ewes on gestation d25 and an ovine luminal epithelial (OLE) cell line shows that histone H3 arginine residues 2, 8, 17 and 26 are citrullinated, but histone H4 arginine 3 is not. Using a pan-PAD inhibitor, we next attenuated histone citrullination in OLE cells, which resulted in a significant decrease in the expression of insulin-like growth factor-binding protein 1 (IGFBP1) mRNA. As IGFBP1 is important for the migration and attachment of the trophectoderm to uterine endometrium, our results suggest that PAD-catalyzed citrullination may be an important post-translational mechanism for the establishment of pregnancy in ewes.

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland.

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 µg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.