HuR drives lung fibroblast differentiation but not metabolic reprogramming in response to TGF-ß and hypoxia.

BACKGROUND: Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-ß1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs). METHODS: Primary HLFs were cultured in the presence (or absence) of TGF-ß1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down. RESULTS: Hypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFß1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-ß1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion. CONCLUSIONS: Hypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-ß. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

Gut Hormones in Health and Obesity: The Upcoming Role of Short Chain Fatty Acids.

We are currently facing an obesity pandemic, with worldwide obesity rates having tripled since 1975. Obesity is one of the main risk factors for the development of non-communicable diseases, which are now the leading cause of death worldwide. This calls for urgent action towards understanding the underlying mechanisms behind the development of obesity as well as developing more effective treatments and interventions. Appetite is carefully regulated in humans via the interaction between the central nervous system and peripheral hormones. This involves a delicate balance in external stimuli, circulating satiating and appetite stimulating hormones, and correct functioning of neuronal signals. Any changes in this equilibrium can lead to an imbalance in energy intake versus expenditure, which often leads to overeating, and potentially weight gain resulting in overweight or obesity. Several lines of research have shown imbalances in gut hormones are found in those who are overweight or obese, which may be contributing to their condition. Therefore, this review examines the evidence for targeting gut hormones in the treatment of obesity by discussing how their dysregulation influences food intake, the potential possibility of altering the circulating levels of these hormones for treating obesity, as well as the role of short chain fatty acids and protein as novel treatments.

Phase II clinical trial of adoptive cell therapy for patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and low-dose interleukin-2.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vß chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).

Regulatory T Cells in Ovarian Cancer Are Characterized by a Highly Activated Phenotype Distinct from that in Melanoma.

Purpose: Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies.Experimental Design: The phenotype of intratumoral and circulating Treg cells was analyzed by multicolor flow cytometry, mass cytometry, RNA-seq, and functional assays.Results: Treg cells isolated from ovarian tumors displayed a distinct cell surface phenotype with increased expression of a number of receptors associated with TCR engagement, including PD-1, 4-1BB, and ICOS. Higher PD-1 and 4-1BB expression was associated with increased responsiveness to further TCR stimulation and increased suppressive capacity, respectively. Transcriptomic and mass cytometry analyses revealed the presence of Treg cell subpopulations and further supported a highly activated state specifically in ovarian tumors. In comparison, Treg cells infiltrating melanomas displayed lower FOXP3, PD-1, 4-1BB, and ICOS expression and were less potent suppressors of CD8 T-cell proliferation.Conclusions: The highly activated phenotype of ovarian tumor-infiltrating Treg cells may be a key component of an immunosuppressive tumor microenvironment. Receptors that are expressed by tumor-infiltrating Treg cells could be exploited for the design of novel combination tumor immunotherapies. Clin Cancer Res; 24(22); 5685-96. ©2018 AACR.

Diversity of Meq gene from clinical Marek's disease virus infection in Saudi Arabia.

AIM: The aim of this study was to demonstrate the genomic features of Meq gene of Marek's disease virus (MDV) recently circulating in Saudi Arabia (SA). MATERIALS AND METHODS: Two poultry flocks suffering from mortalities and visceral tumors were presented to the Veterinary Teaching Hospital, King Faisal University, SA. Subjected to different diagnostic procedures: Case history, clinical signs, and necropsy as well as polymerase chain reaction followed by Meq gene sequence analysis. RESULTS: Case history, clinical signs, and necropsy were suggestive of MDV infection. The Meq gene was successfully detected in liver and spleen of infected chickens. A 1062 bp band including the native Meq ORF in addition to a 939 bp of S-Meq (short isoform of Meq) were amplified from Saudi 01-13 and Saudi 02-13, respectively. The nucleotide and deduced amino acids sequences of the amplified Meq genes of both Saudi isolates showed distinct polymorphism when compared with the standard USA virulent isolates Md5 and GA. The sequence analysis of the S-Meq gene showed a 123 bp deletion representing 41 amino acids between two proline-rich areas without any frameshift. The Meq gene encoded four repeats of proline-rich repeats (PRRs sequences), whereas the S-Meq contains only two PRRs. Interestingly, the phylogenetic analysis revealed that both of SA MDV isolates are closely related to the MDV strains from Poland. CONCLUSION: The two MDV isolates contain several nucleotide polymorphisms resulting in distinct amino acid substitutions. It is suggested that migratory and wild birds, as well as world trading of poultry and its by-products, have a great contribution in the transmission of MDVs overseas.

Gene of the month: BAP1.

The BAP1 gene (BRCA1-associated protein 1) is a tumour suppressor gene that encodes a deubiquitinating enzyme (DUB), regulating key cellular pathways, including cell cycle, cellular differentiation, transcription and DNA damage response. Germline BAP1 mutations cause a novel cancer syndrome characterised by early onset of multiple atypical Spitz tumours and increased risk of uveal and cutaneous melanoma, mesothelioma, renal cell carcinoma and various other malignancies. Recognising the clinicopathological features of specific BAP1-deficient tumours is crucial for early screening/tumour detection, with significant impact on patient outcome.

Eosinophilic Fasciitis and Acute Encephalopathy Toxicity from Pembrolizumab Treatment of a Patient with Metastatic Melanoma.

Anti-PD-1 inhibitors have significant activity in metastatic melanoma. Responses often occur early and may be sustained. The optimal duration of treatment with these agents is unknown. Here, we report the case of a 51-year-old woman treated with pembrolizumab, as part of the Keynote-001 trial, as first-line treatment for metastatic disease. She experienced a complete response after 13.8 months of treatment with no adverse events. One month after the last drug infusion and 18 months from starting treatment, the patient presented with eosinophilic fasciitis. She then developed acute confusion and weakness, thought to be due to intracranial vasculitis. High-dose steroids were initiated with resolution of the fasciitis. Aspirin was commenced for presumed vasculitis with resolution of the neurologic symptoms. To our knowledge, there are no previous reports of eosinophilic fasciitis or cerebral vasculitis due to anti-PD-1 agents. This case demonstrates that toxicity may occur in association with pembrolizumab treatment after a prolonged period of treatment without toxicity. Future trials should explore the optimal duration of treatment with pembrolizumab.

Deletion of low molecular weight protein tyrosine phosphatase (Acp1) protects against stress-induced cardiomyopathy.

The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, is a ubiquitously expressed phosphatase whose in vivo function in the heart and in cardiac diseases remains unknown. To investigate the in vivo role of LMPTP in cardiac function, we generated mice with genetic inactivation of the Acp1 locus and studied their response to long-term pressure overload. Acp1(-/-) mice develop normally and ageing mice do not show pathology in major tissues under basal conditions. However, Acp1(-/-) mice are strikingly resistant to pressure overload hypertrophy and heart failure. Lmptp expression is high in the embryonic mouse heart, decreased in the postnatal stage, and increased in the adult mouse failing heart. We also show that LMPTP expression increases in end-stage heart failure in humans. Consistent with their protected phenotype, Acp1(-/-) mice subjected to pressure overload hypertrophy have attenuated fibrosis and decreased expression of fibrotic genes. Transcriptional profiling and analysis of molecular signalling show that the resistance of Acp1(-/-) mice to pathological cardiac stress correlates with marginal re-expression of fetal cardiac genes, increased insulin receptor beta phosphorylation, as well as PKA and ephrin receptor expression, and inactivation of the CaMKIIδ pathway. Our data show that ablation of Lmptp inhibits pathological cardiac remodelling and suggest that inhibition of LMPTP may be of therapeutic relevance for the treatment of human heart failure.

Utility and diagnostic accuracy of endobronchial ultrasound-guided transbronchial fine-needle aspiration cytology of mediastinal lesions: Saudi Arabian experience.

OBJECTIVE: The objective of this study is to evaluate the cytological accuracy of endobronchial ultrasound-guided transbronchial fine-needle aspiration (EBUS-TFNA) of the mediastinal mass/nodular lesions. STUDY DESIGN: Over 3½ years from inception at King Khalid University Hospital, a retrospective analysis of the cytological diagnoses of all the EBUS-TFNA procedures performed in 80 patients who had mediastinal mass/nodular enlargement. Cytology results were reviewed and correlated with the histologic follow-up. RESULTS: Of the 80 patients who underwent EBUS-TFNA, 15 cases (18.75%) were positive for malignancy, 48 cases (60%) negative for malignancy and 17 cases (21.25%) unsatisfactory. Of the 48 cases, which were negative for malignancy, 24 (50%) cases were of granulomatous inflammation. The overall diagnostic yield of our EBUS-TFNA specimen was 78.75%. Forty-seven cases (58.75%) of 80 cases had histological follow-up biopsies. Among them, 32 cases (68%) had the same cytological and histological diagnosis and 15 cases (31.09%) had discordance between the cytology and the follow-up histological diagnosis. The sensitivity, specificity, and positive and negative predictive values for diagnosing granulomas by EBUS-TFNA are 77%, 82%, 83%, and 75% and for diagnosing malignancy are 71%, 100%, 100%, and 82%, respectively. CONCLUSION: Preliminary results show that cytological samples obtained through EBUS-TFNA are accurate and specific in making a diagnosis of the mediastinal mass/nodular lesions. Its optimum use depends on the effective collaboration between the cytotechnologist, pathologist, and the bronchoscopist.

Assessment of suicidal and self-injurious behaviours among patients with depression.

The aim of this cross-sectional 1-year study was to estimate the prevalence and to identify the risk factors for suicidal and self-injurious behaviours among patients with depression. A convenience sample of 557 out- and inpatients diagnosed with mood disorders was selected from 3 treatment settings in Riyadh, Saudi Arabia. Eligible patients completed data on sociodemographic variables and the Columbia suicide severity rating scale risk assessment version. The reported prevalence of attempted suicide in the previous week was 36.6%, interrupted suicide attempt 29.8%, aborted suicide attempt 34.6% and self-injurious behaviour without suicide intent 7.7%. Concerning suicide ideation, 47.2% reported suicidal thoughts, 36.6% suicidal thoughts with methods but without a specific plan and 35.4% suicidal intent without a specific plan. Male sex, joint family type, literate education, being in employment, smoking and physical co-morbidities were significantly associated with all types of suicide behaviour.

Career characteristics and postgraduate education of female dentist graduates of the College of Dentistry at King Saud University, Saudi Arabia.

OBJECTIVES: The aims of this study were to investigate the number of female bachelor of dental surgery graduates who earned postgraduate education degrees from King Saud University, their specialties, degrees of qualification, and countries of graduation. METHODS: A questionnaire survey was conducted through face-to-face or phone interview among 677 female dentists who graduated from the College of Dentistry at King Saud University between 1984 and 2006. RESULTS: Five hundred forty-five (81%) graduate female dentists responded, of whom 54% had completed postgraduate dental education. In the Saudi Board, 17% had obtained degrees in advanced restorative dentistry, 15% in pediatric dentistry, 14% in advanced general dentistry, 11% in orthodontics, 10% in prosthodontics, 8% in operative dentistry, 5% in endodontics, 5% in periodontics, 3% in oral and maxillofacial surgery, and 11% in other specialties. Fifty-nine percent had master's degrees, 7% had doctorates, 32% had board certificates, and 2% had fellowship certificates. CONCLUSION: More than half (54%) the female dentists surveyed had pursued postgraduate education. This investigation highlighted the different specialties enrolled by female dentists. Orthodontic post graduate program displayed the highest percentage of interest. Understanding the unavoidable social family needs and the demand for higher education by female dentists provides ideas as more programs should be planned to accommodate the needs of Saudi female dentists. This type of study should be repeated due to the increasing interest of female dentists in higher education.

Syringoid eccrine carcinoma: a clinicopathological and immunohistochemical study of four cases.

BACKGROUND: Syringoid eccrine carcinoma (SEC) is a rare malignant adnexal tumour with variable presentations. AIM: To examine the clinicopathological and immunohistochemical features of SEC. METHODS: Four cases were reviewed by three dermatopathologists and the immunohistochemical profile was examined using antibodies against CK5/6, CK7, CK14, CK20, LMWK, HMWK, EMA, mCEA, p63, ER, PR, AR, S-100 and Ber-EP4. RESULTS: The cases occurred in two men and two women, ranging in age from 61 to 87 years (mean 68.5). Two of the lesions were from the face and two from the trunk. All four lesions were composed of an atypical infiltrative mass with syringoma-like tadpole morphology with ductular differentiation and prominent desmoplasia. Three cases demonstrated perineural invasion and two had positive lymph node metastases. Immunostaining was variable. Immunohistochemistry positivity was as follows: three out of four cases were positive for CK5/6, CK7 (2/4), CK14 (1/3), CK20 (0/2), HMWK (0/2), LMWK (1/2), EMA (3/4), mCEA (4/4), p63 (2/3), ER (2/3), PR (1/2), AR (0/3), S-100 (0/3) and Ber-EP4 (2/2). CONCLUSION: SEC can present on the trunk and are not limited to the head and neck region. In addition to syringoma-like tadpole structures and glandular differentiation, these tumours can also exhibit squamoid and cribriform growth patterns. Immunostaining in SEC is variable and this variability is believed to stem from this tumour's ability to differentiate along multiple routes, including sweat secretory and/or ductal differentiation.

Expansion and characterization of human melanoma tumor-infiltrating lymphocytes (TILs).

BACKGROUND: Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution. PRINCIPAL FINDINGS: TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days. CONCLUSIONS: TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT.

Mental and Social Health Atlas I in Saudi Arabia: 2007-08.

Based on the World Health Organization's Mental Health Atlas, the first Mental and Social Health Atlas in Saudi Arabia describes the historical background of mental health and social services in the country and identifies several deficiencies in the system including infrastructure and logistics and lack of epidemiological data. There is now great progress in strategic planning for developing and improving mental health care services across the nation, with suggestions to develop psychiatric services for identified special populations, to establish community mental health care services, to improve research and training in mental health, and to update mental health annual information systems using advanced information technology.

Benign mixed tumor of the skin, hypercellular variant: a case report.

A 71-year-old man presented with a slowly growing 2.0x2.0x1.0 cm scalp lesion that was surgically removed. Microscopic examination showed a well-circumscribed dermally located tumor composed of ductal elements lined by double to multiple cell layers of bland cuboidal inner cells and elongated spindled outer cells with areas showing cribriform and solid growth patterns. Some cells showed prominent cytoplasmic clearing. A few mitotic figures are noted ranging from 1-2 mitotic figure/10 hpf. There are also foci of squamous differentiation as well as occasional mature adipocytes. The background stroma was predominantly sclerotic with only small area of myxoid background (confirmed by Hale's colloidal iron). Immunohistochemical studies revealed positive immunoreactivity for EMA, CEA, CD117, HWMK, LWMK, CK7, Androgen receptor and S100 in the ductal (epithelial) cells and positive immunereactivity for calponin, SMA, CK 5/6 and p63 in the myoepithelial component. No immunoreactivity for Brst-2, ER, PR and CK20 was noted. MIB-1 showed mildly increased proliferrative index highlighting 5% of the nuclei. The overall morphology and immunohistochemical profile are that of a benign cutanoues mixed tumor (chondroid syringoma). Given the unusual striking celluarlity, we suggest to subclassify this as a hyper-cellular variant.

Exertional dizziness and syncope caused by anomalous left coronary artery origin from the right sinus of Valsalva.

Coronary anomalies are generally rare, but has fascinating congenital cardiac disease entities, recognized in less than 1.3% of all coronary angiograms. Left coronary artery arising from right sinus of valsalva RSOV represents an extremely uncommon subtype. Initial presentations include chest pain, myocardial infarction, arrhythmias, sudden death, and rarely exertional syncope. We report a case of exertional dizziness and syncope, diagnosed to have anomalous origin of left main coronary artery from RSOV. Surgical intervention was curative.

Rescue in situ thrombolysis for acute coronary thromboembolism in an angiographically normal coronary artery.

Coronary thromboembolism in an angiographically normal coronary artery is extremely uncommon. There are few instances where normal coronary arteries have been documented just prior to an episode of acute thromboembolic insult. We now report such a case of acute coronary thromboembolism in a patient with widely patent coronary vessels documented just prior to the event during preoperative screening angiogram with successful in situ revascularization.

Smoking patterns among primary health care attendees, Al-Qassim region, Saudi Arabia.

Prevalence, sociodemographic patterns and medical co-morbidity of smoking among a cross-section of primary health care (PHC) clients in Saudi Arabia were examined. We used a 44-item semistructured questionnaire to collect data from 1752 patients at 25 randomly selected PHC centres. Percentage of smoking was 52.3%. Although 85% were adult smokers, 8.6% began smoking before age 12. Smokers gave overlapping reasons to smoke including peer pressure; non-smokers gave religious and health logics against smoking. Of all smokers, 92.8% wanted to learn cessation strategies, 11.8% were ignorant of hazards and 32.4% reported manifestations of nicotine withdrawal. Besides alcohol use (13.4%), 81.8% had co-morbid physical disease.