Pilonidal sinus laser-assisted closure (PiLAC) - a low-morbidity alternative to excision with excellent long-term outcomes.

INTRODUCTION: Recurrence after surgery for pilonidal sinus disease is a recognised problem and patients often re-present months after discharge. We routinely treat primary and recurrent pilonidal sinus disease with Pilonidal sinus Laser-Assisted Closure (PiLAC). Long-term outcomes following PiLAC surgery was examined following clinical and telephone review. METHODS: All patients undergoing PiLAC as a day-case between April 2016 and July 2019 were included. Patients were followed up in a nurse-led clinic until complete healing or recurrence. A prospective database and retrospective audit of notes combined with longer-term follow-up by telephone were used. RESULTS: A total of 35 patients underwent PiLAC, median age 28 (18-53 years), 28 males:7 females. A total of 28 patients had long-term (>60 days) follow-up, mean 407 days (range 67-887 days); 25/28 patients (89.3%) had healed with no recurrence on long-term follow-up. Of these 28 patients, 11 were first presentation of pilonidal disease and underwent PiLAC as their first treatment, with a 91% heal rate long term. A total of 15 patients had seton drainage prior to PiLAC, with a 93% heal rate versus no seton (83%). Fisher's exact test showed no significant difference between sex, new/recurrent pilonidal disease and seton placement (p>0.05). CONCLUSIONS: Healing after PiLAC for the treatment of primary and recurrent pilonidal sinus disease is preserved with excellent long-term outcomes. We recommend it as an alternative to surgical excision.

Leveraging Genomics for Head and Neck Cancer Treatment.

The genomic landscape of head and neck squamous cell carcinoma (HNSCC) has been recently elucidated. Key epigenetic and genetic characteristics of this cancer have been reported and substantiated in multiple data sets, including those distinctive to the growing subset of human papilloma virus (HPV)-associated tumors. This increased understanding of the molecular underpinnings of HNSCC has not resulted in new approaches to treatment. Three Food and Drug Administration-approved molecular targeting agents are currently available to treat recurrent/metastatic disease, but these have exhibited efficacy only in subsets of HNSCC patients, and thus surgery, chemotherapy, and/or radiation remain as standard approaches. The lack of predictive biomarkers to any therapy represents an obstacle to achieving the promise of precision medicine. This review aims to familiarize the reader with current insights into the HNSCC genomic landscape, discuss the currently approved and promising molecular targeting agents under exploration in laboratories and clinics, and consider precision medicine approaches to HNSCC.

Frequent promoter hypermethylation of PTPRT increases STAT3 activation and sensitivity to STAT3 inhibition in head and neck cancer.

Signal transducer and activator of transcription 3 (STAT3) overactivation is a common event in many cancers, including head and neck squamous cell carcinoma (HNSCC), where STAT3 represents a promising therapeutic target. HNSCC is not characterized by frequent kinase mutations, in contrast to some malignancies where mutational activation of kinases upstream of STAT3 is common. Instead, STAT3 may be activated by loss-of-function of negative regulators of STAT3, including by promoter hypermethylation of PTPRT. Here we first analyzed The Cancer Genome Atlas data and determined that the PTPRT promoter is frequently hypermethylated in several cancers, including HNSCC (60.1% of tumors analyzed) in association with downregulation of PTPRT mRNA expression and upregulation of pSTAT3 expression. These findings were confirmed in an independent cohort of HNSCC tumors by methylation-specific PCR and immunohistochemistry. We demonstrate that PTPRT promoter methylation and gene silencing is reversible in HNSCC cells, leading to PTPRT-specific downregulation of pSTAT3 expression. We further show that PTPRT promoter methylation is significantly associated with sensitivity to STAT3 inhibition in HNSCC cells, suggesting that PTPRT promoter methylation may serve as a predictive biomarker for responsiveness to STAT3 inhibitors in clinical development.

You eat what you are: autophagy inhibition as a therapeutic strategy in leukemia.

A deeper understanding of the role of autophagy, literally 'self-eating', in normal and cancer cell biology has emerged over the last few years. Autophagy serves as a vehicle for cells to respond to various stressors including genomic, hypoxic and nutrient stress, and to oppose mechanisms of 'programmed' cell death. Here, we review not only mechanisms of cell death and cell survival but also the early successes in applying autophagy inhibition strategies in solid tumors using the only currently available clinical inhibitor, oral hydroxychloroquine. In acute leukemia, currently available chemotherapy drugs promote cell death and demonstrate clinical benefit, but relapse and subsequent chemotherapy resistance is common. Increasing preclinical data suggest that autophagy is active in leukemia as a means of promoting cell survival in response to chemotherapy. We propose coupling autophagy inhibition strategies with current cytotoxic chemotherapy and discuss synergistic combinations of available anti-leukemic therapies with autophagy inhibition. Furthermore, novel autophagy inhibitors are in development and promise to provide new therapeutic opportunities for patients with leukemia.

DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies.

The development of effective treatment strategies for most forms of acute myeloid leukemia (AML) has languished for the past several decades. There are a number of reasons for this, but key among them is the considerable heterogeneity of this disease and the paucity of molecular markers that can be used to predict clinical outcomes and responsiveness to different therapies. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatment that are based on individual mutational profiles. It is particularly notable that studies by The Cancer Genome Atlas and others have determined that 44% of patients with AML exhibit mutations in genes that regulate methylation of genomic DNA. In particular, frequent mutation has been observed in the genes encoding DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), as well as Tet oncogene family member 2. This review will summarize the incidence of these mutations, their impact on biochemical functions including epigenetic modification of genomic DNA and their potential usefulness as prognostic indicators. Importantly, the presence of DNMT3A, IDH1 or IDH2 mutations may confer sensitivity to novel therapeutic approaches, including the use of demethylating agents. Therefore, the clinical experience with decitabine and azacitidine in the treatment of patients harboring these mutations will be reviewed. Overall, we propose that understanding the role of these mutations in AML biology will lead to more rational therapeutic approaches targeting molecularly defined subtypes of the disease.

STAT transcription factors in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention.

Signal transducer and activator of transcription (STAT) proteins comprise a family of transcription factors that are activated by cytokines, hormones and growth factors. The activation of STAT proteins plays a key role in the production of mature hematopoietic cells via effects on cellular proliferation, survival and lineage-specific differentiation. Emerging evidence also demonstrates frequent, constitutive activation of STATs in primary leukemia specimens. Moreover, roles for STATs in promoting leukemia development have been delineated in numerous preclinical studies. This review summarizes our current understanding of STAT protein involvement in normal hematopoiesis and leukemogenesis, as well as recent advances in the development and testing of novel STAT inhibitors.

A robust MRI-compatible system to facilitate highly accurate stereotactic administration of therapeutic agents to targets within the brain of a large animal model.

Achieving accurate intracranial electrode or catheter placement is critical in clinical practice in order to maximise the efficacy of deep brain stimulation and drug delivery respectively as well as to minimise side-effects. We have developed a highly accurate and robust method for MRI-guided, stereotactic delivery of catheters and electrodes to deep target structures in the brain of pigs. This study outlines the development of this equipment and animal model. Specifically this system enables reliable head immobilisation, acquisition of high-resolution MR images, precise co-registration of MRI and stereotactic spaces and overall rigidity to facilitate accurate burr hole-generation and catheter implantation. To demonstrate the utility of this system, in this study a total of twelve catheters were implanted into the putamen of six Large White Landrace pigs. All implants were accurately placed into the putamen. Target accuracy had a mean Euclidean distance of 0.623 mm (standard deviation of 0.33 mm). This method has allowed us to accurately insert fine cannulae, suitable for the administration of therapeutic agents by convection-enhanced delivery (CED), into the brain of pigs. This study provides summary evidence of a robust system for catheter implantation into the brain of a large animal model. We are currently using this stereotactic system, implantation procedure and animal model to develop catheter-based drug delivery systems that will be translated into human clinical trials, as well as to model the distribution of therapeutic agents administered by CED over large volumes of brain.

Effects of ractopamine and sex on serum metabolites and skeletal muscle gene expression in finishing steers and heifers.

We evaluated growth-related responses to ractopamine in steers and heifers. Sixteen Angus steers (512 kg) and 16 Angus heifers (473 kg) housed in individual pens were used in a complete block design. At 90 to 97 d before the experiment, steers were implanted with 120 mg of trenbolone acetate and 24 mg of estradiol-17beta (Component TE-S) and heifers were implanted with 140 mg of trenbolone acetate and 14 mg of estradiol-17beta (Component TE-H). Treatments were arranged as a 2 x 2 factorial and included sex (steer vs. heifer) and ractopamine-HCl (0 or 200 mg/d). Cattle were fed a diet based on steam-flaked corn once daily. Blood and LM and biceps femoris (BF) biopsy samples were collected on d 0 (before ractopamine feeding) and after 14 and 28 d of ractopamine feeding. Serum insulin concentrations were not affected by ractopamine or sex. Serum IGF-I concentrations were greater in steers than heifers (P < 0.001), and steers demonstrated greater IGF-I mRNA expression in BF than heifers (P = 0.05). Ractopamine decreased serum IGF-I concentrations in heifers on d 14, but increased serum IGF-I concentrations in steers on d 28 (sex x ractopamine x day interaction; P = 0.03). Ractopamine did not affect (P >or= 0.19) mRNA expression of IGF-I, IGFBP-3, or calpastatin in BF or LM. However, ractopamine led to increases in LM expression of IGFBP-5 in heifers, but to decreases in expression in steers (ractopamine x sex interaction; P = 0.04). Ractopamine decreased myosin heavy chain IIA mRNA expression in BF (P = 0.04) but not in LM (P = 0.99). Ractopamine decreased beta(2)-receptor mRNA expression in LM of steers on d 14, but not on d 28; in contrast, expression of beta(2)-receptor mRNA in LM of heifers was not affected by ractopamine (sex x ractopamine x day interaction; P = 0.03). Although there were a few criteria for which ractopamine led to differences in response between steers and heifers, there were no striking disparities to suggest that the effectiveness of ractopamine would markedly differ between sexes.

Plant folk medicines for gastrointestinal disorders among the main tribes of Sonora, Mexico.

This paper describes the herbal remedies used by ethnic groups from Sonora, Mexico, for treatment of gastrointestinal diseases. Twelve types of these illnesses are cured using 85 different species which belong to 38 families. Thirty nine spp. are used to treat diarrhea, 28 for stomach-ache, 12 for constipation, 9 for intestinal parasites, 6 for indigestion, 3 for stomach or intestinal cancer, 3 for stomach inflammation and only 1 to treat gastrointestinal sicknesses, ulcers, gastritis, colitis and colic. Regarding the use of species of plant per ethnic group the following was observed: Mayo 47; Seri, 27; Yaqui, 13; Guarijio, 12, Pima, 5 and Papago, 3. The plants are used by two or more tribes, for the same or different illness but always related to the gastrointestinal system.

Signal transduction pathways that contribute to myeloid differentiation.

The production of mature, differentiated myeloid cells is regulated by the action of hematopoietic cytokines on progenitor cells in the bone marrow. Cytokines drive the process of myeloid differentiation by binding to specific cell-surface receptors in a stage- and lineage-specific manner. Following the binding of a cytokine to its cognate receptor, intracellular signal-transduction pathways become activated that facilitate the myeloid differentiation process. These intracellular signaling pathways may promote myelopoiesis by stimulating expansion of a progenitor pool, supporting cellular survival during the differentiation process, or by directly driving the phenotypic changes associated with differentiation. Ultimately, pathways that drive the differentiation process converge on myeloid transcription factors, including PU.1 and the C/EBP family, that are critical for differentiation to proceed. While much is known about the cytokines, cytokine receptors and transcription factors that regulate myeloid differentiation, less is known about the precise roles that specific signaling mediators play in promoting myeloid differentiation. Recently, however, the application of novel pharmacologic inhibitors, siRNA strategies, and transgenic and knockout models has begun to shed light on the involvement and function of signaling pathways in normal myeloid differentiation. This review will discuss the roles that key signaling pathways and mediators play in myeloid differentiation.

Effect of glycine and vitamin supplementation on sulphur amino acid utilization by growing cattle.

Effects of glycine (Gly) and B-vitamins on sulphur amino acid (AA) utilization were studied in growing steers maintained under conditions where methionine (Met) was first limiting. Conditions were generated by limit feeding a diet low in ruminally non-degraded protein and abomasally infusing an AA mixture limiting in Met. Retained N tended (p = 0.07) to improve when steers received 10 mg folate, 10 mg vitamin B6, and 0.10 mg vitamin B12 daily. Hepatic vitamin B12 (p = 0.08) and folate (p = 0.05) concentrations increased with vitamin supplementation. In another trial, factorial treatments were 2 or 5 g/day L-Met and 0 or 50 g/day Gly infused abomasally. Retained N increased (p < 0.05) in response to Met, and responses were numerically larger in the presence of supplemental Gly. In a different trial, factorial treatments were 0 or 2.4 g/day L-cysteine (Cys) and 0 or 40 g/day Gly. Retained N was not affected by Cys in the absence of Gly, but was increased by Cys when Gly was supplemented (interaction, p = 0.01). B-vitamin status may affect sparing of Met by Cys. Supplemental Gly improved responses to supplemental Met and Cys.

Decreased prostatic arachidonic acid in human prostatic carcinoma.

OBJECTIVE: To measure prostatic and blood fatty acid composition in a large group of patients undergoing prostatectomy for benign or malignant prostate disease, as there is evidence linking arachidonic acid metabolism and prostate cancer through its role as an eicosanoid precursor, and earlier studies showed lower prostatic arachidonic acid content in a few patients. PATIENTS AND METHODS: Prostatic phospholipid fatty acid composition was determined in prostate tissue from 173 patients undergoing prostate surgery, i.e. radical prostatectomy, cystoprostatectomy or transurethral resection (TURP). Blood fatty acid composition was determined in 99 of these patients and in 85 undergoing prostatic needle biopsy. RESULTS: There was a significantly lower percentage of arachidonic acid in malignant than in benign portions of the prostate (15.2% vs 17%) in all patients assessed. The changes were greatest in those undergoing TURP for known prostate cancer (13.4% vs 17.2%), these patients having the greatest proportion of malignancy in the specimens. There were no consistent changes in blood fatty acid composition. CONCLUSION: This is the first prospective study of arachidonic acids levels involving many consecutive patients undergoing prostate surgery for either benign or malignant disease. The lower prostatic arachidonic acid level is probably a result of the increased use of arachidonic acid for producing prostaglandins and/or leukotrienes. Further understanding of the cause and/or consequence of this finding might lead to a better understanding of prostate cancer.

Recent advances in the development of anticancer agents targeting cell death inhibitors in the Bcl-2 protein family.

Hematopoietic malignancies frequently are characterized by defects in apoptosis signaling. This renders the malignant cells resistant to endogenous apoptotic stimuli, as well as exogenous stimuli, such as chemotherapy drugs and radiation. The defective apoptosis seen in human cancers often results from overexpression of antiapoptotic proteins in the Bcl-2 protein family, particularly Bcl-2 and Bcl-X(L). A great deal of effort is currently aimed at developing novel agents to inhibit the expression or function of these proteins. Antisense agents directed against Bcl-2 mRNA are showing considerable promise in clinical trials. In addition, detailed knowledge of the structures of Bcl-2 and Bcl-X(L), coupled with high-throughput and computer-assisted screening of chemical libraries, has led to the identification of a number of short peptides and small organic molecules capable of inhibiting Bcl-2 and Bcl-X(L) function. These newly described agents hold considerable promise for enhancing the chemo- and radiation sensitivities of Bcl-2- and Bcl-X(L)-overexpressing cancers. This review will highlight recent advances in the development and testing of agents targeting cell death inhibitors in the Bcl-2 protein family.

Differential activation of apoptosis regulatory pathways during monocytic vs granulocytic differentiation: a requirement for Bcl-X(L)and XIAP in the prolonged survival of monocytic cells.

Neutrophils and monocytes/macrophages are derived from common progenitors, but exhibit markedly different lifespans. Differentiated neutrophils are short-lived and die rapidly by apoptosis, while monocytic cells are longer-lived. In this report we used the HL-60 cell line as a model system to identify differences in apoptotic pathways which might account for the differing lifespans of granulocytic vs monocytic cells. We observed that induction of granulocytic differentiation by retinoic acid led to robust activation of the executioner protease caspase-3, and early onset of apoptosis. By contrast, caspase-3 was not appreciably activated during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation, and apoptosis was delayed in these cells. Since the activation of caspase-3 is inhibited by members of the inhibitor of apoptosis (IAP) and Bcl-2 protein families, we investigated the expression of anti-apoptotic members of these families. Induction of monocytic differentiation led to marked upregulation of the IAP protein XIAP, as well as the Bcl-2 family member Bcl-X(L). During granulocytic differentiation the levels of XIAP progressively declined, while Bcl-X(L) levels remained unchanged. A different IAP protein, survivin, was downregulated during differentiation along either lineage, as was expression of Bcl-2. The upregulation of Bcl-X(L) during monocytic differentiation coincided with phosphorylation/activation of STAT3, a known activator of bcl-X gene transcription. Moreover, Bcl-X(L) upregulation was dependent on MEK/ERK signaling. Upregulation of XIAP proceeded in a MEK/ERK-independent fashion. Treatment with antisense Bcl-X(L) or XIAP oligonucleotides resulted in significant loss of viability in cells differentiating along the monocytic lineage. Together, these findings indicate that the levels of XIAP and Bcl-X(L) are regulated by distinct pathways during monocytic differentiation, and that upregulation of these proteins contributes to the increased longevity of cells in the monocytic lineage.

Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines.

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.

Interferon alpha2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers.

A recombinant adenovirus expressing human interferon alpha2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells.

Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus.

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.