Development and operation of the defence COVID-19 lab as a SARS-CoV-2 diagnostic screening capability for UK military personnel.

BACKGROUND: In the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel. LABORATORY STRUCTURE AND RESOURCE: The DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists. LABORATORY RESULTS: Over 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started. CONCLUSIONS: Through a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

Clinical implications of neuroendocrine differentiation in prostate cancer.

The cellular signaling pathways of the prostate play a central role in the induction, maintenance, and progression of prostate cancer (CaP). Neuroendocrine (NE) cells demonstrate attributes that suggest they are an integral part of these signaling cascades. We summarize what is known regarding NE cells in CaP focusing on NE cellular transdifferentiation. This significant event in CaP progression appears to be accelerated by androgen deprivation (AD) treatment. We examine biochemical pathways that may impact NE differentiation in a chronological manner focusing on AD therapy (ADT) as a central event in inducing androgen-independent CaP. Our analysis is limited to the common adenocarcinoma pattern of CaP and excludes small-cell and carcinoid prostatic variants. In conclusion, we speculate on the future of treatment and research in this area.

Inhibition of prostate cancer neovascularization and growth by urokinase-plasminogen activator receptor blockade.

Binding of the serine protease urokinase (u-PA) to its receptor on tumor cell surfaces facilitates proteolysis and tumor invasion. We undertook this study to determine whether the role of u-PA in prostate cancer induced angiogenesis and secondary tumor growth by developing a homologous, immunocompetent in vivo model in which the tumors cells secrete an inhibitor of the murine u-PA receptor. A mutant recombinant murine u-PA that retains receptor binding but not proteolytic activity was made by PCR mutagenesis. Mutant u-PA and a reporter gene pRK luciferase were transfected and stably expressed in the highly metastatic rat Dunning MAT-LyLu prostate cancer cell line. Several clones expressing mutant u-PA and luciferase were identified by Western blotting, plasminogen zymography, and reverse transcription-PCR. One of these clones, 5C4, was injected s.c. into Copenhagen rats. Compared to animals injected with clones expressing pRK luciferase alone, tumors in animals injected with 5C4 cells were significantly smaller. Moreover, there were fewer lung micrometastases in the 5C4 animals. Primary tumor angiogenesis was measured by microvessel quantification of tissue stained with antibodies against von Willebrand factor. Mean microvessel density in 5C4 tumors was 4.3-fold lower than that in animals with tumors derived from the control tumor cell line (P < 0.0001). Significant inhibition of tumor growth was also observed for two additional MAT-LyLu cell lines expressing mutant u-PA. These findings suggest that cell surface u-PA contributes to prostate cancer growth by enhancing angiogenesis.

Extrathoracic localization of Legionella pneumophila in Legionnaires' pneumonia.

Extrathoracic organs of 12 fatal cases of Legionnaires' disease from the 1977 Vermont epidemic were reviewed for the presence of Legionella pneumophila. The bacteria were identified by direct immunofluorescence microscopy of paraffin-embedded spleen, liver, lymph node, and kidney tissue. Extrathoracic bacteria were demonstrated in six of the 12 cases (3/7 spleens, 2/9 livers, and 2/10 kidneys). In the liver and spleen, the legionellae were associated with cells of the reticuloendothelial system. In the kidney, the bacteria were restricted to the interstitium and renal tubules. There were no acute inflammatory lesions in any of these organs. The distribution of bacteria in the extrathoracic viscera is compatible with dissemination through the blood stream.

Decreased prostate cancer cell migration by inhibition of the insulin-like growth factor II/Mannose-6-Phosphate receptor.

The 270-kDa insulin-like growth factor II (IGF-II)/cation-independent mannose-6-phosphate receptor (MPR) is a multifunctional receptor protein. Endocytoses and intracellular transport of soluble enzymes bearing mannose6-phosphate (M-6-P) residues to lysosomes is mediated by the IGF-II/MPR. We examined human prostate cancer cells for IGF-II/MPR expression to determine whether this receptor mediates cell migration. PC3 human prostate cancer cells were studied for intracellular IGF-II/MPR by immunoblotting. PC3 cell surface IGF-II/MPR expression was assessed by flow cytometric analysis. Cell motility was quantitated by a scratch migration assay, and IGF-II/MPR blockade was achieved using M-6-P or affinity-purified rabbit anti-bovine cation-independent IGF-II/MPR immunoglobulin. IGF-II/MPR is expressed in the cytoplasm and on the surface of PC3 prostate cancer cells. The mean number of PC3 cells migrating per high powered field in medium containing polyclonal anti-IGF-II/MPR immunoglobulin or M-6-P decreased significantly (5 ± 4 cells and 34 ± 5 cells, respectively) compared with control medium containing mouse immunoglobulin G (70 ± 12 cells) or mannose-1-phosphate (67 ± 7 cells). This decreased PC3 cell migration following cell surface IGF-II/MPR blockade suggests that the IGF-II/MPR may play an important role in prostate cancer cell motility.

Determining variables for repeat prostate biopsy.

During the evaluation of prostate cancer, men who have undergone transrectal ultrasound-guided biopsy with negative results present a dilemma as to what further follow-up is required. Multiple variables have been proposed throughout the literature to improve cancer detection rates not only in initial biopsy results, but also on repeat evaluation. These variables include prostate-specific antigen (PSA) velocity, PSA density, free-percent PSA, and histological findings, each of which may singly or collectively dictate the need for further biopsy. After performing a Medline literature search using specific Medical Subject Headings (prostate biopsy, repeat prostate biopsy, PSA velocity, PSA density, free-percent PSA, prostate inflammation), we critically evaluated pertinent articles. Using this accumulated data and information, we composed an algorithm to assist in the decision process for repeat biopsy.

Management of prostatitis.

Prostatitis is a common clinical entity with a prevalence rate of 5-9% and accounts for over 2 million hospital visits annually in the USA. It is traditionally classified into acute bacterial, chronic bacterial, abacterial prostatitis and prostatodynia. The recent consensus conference of the US National Institute of Diabetes and Digestive and Kidney Diseases in 2000 resulted in renewed interest in the prevalence, etiology, pathogenesis and treatment of the prostatitis syndromes. In this review, we present the contemporary knowledge and experience regarding the etiology, classification, evaluation and treatment of this condition including the role of transurethral microwave hyperthermia and transurethral needle ablation.

Can single dose preoperative intrathecal morphine sulfate provide cost-effective postoperative analgesia and patient satisfaction during radical prostatectomy in the current era of cost containment?

We retrospectively analyzed the analgesic efficacy and surgical outcomes of a single preoperative intrathecal long-acting morphine sulfate injection (0.25-0.5 mg) and postoperative intravenous (i.v.) ketorolac in 62 patients who underwent radical retropubic prostatectomy (RRP). Total postoperative analgesic requirement was documented along with assessment of length of hospital stay, pain control and time for resumption of normal activity. Postoperatively, 45% of patients required only nonsteroidal agents (ketorolac), whereas 55% needed a mean of 13.3 mg of supplemental i.v. morphine sulfate. Mean hospital stay was 2.3+/-0.3 days. Eighty-two per cent of patients felt the length of hospital stay adequate. Ninety-seven per cent of patients were satisfied with anesthesia selected and 95% of patients considered pain control on postoperative days 1 and 2 as effective. All patients resumed to full physical activity by 5.3+/-0.4 weeks after surgery. We conclude that a single preoperative injection of intrathecal morphine sulfate combined with i.v. ketorolac postoperatively results in effective analgesia, diminished supplemental narcotic requirement and high patient satisfaction during radical retropubic prostatectomy.

Elements regulating angiogenesis and correlative microvessel density in benign hyperplastic and malignant prostate tissue.

PURPOSE: To quantify the ex vivo production of proangiogenic proteins (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (tPA)) and angiogenesis inhibitors (plasminogen activator inhibitor type-1 (PAI-1) and angiostatin) from epithelial and stromal components of primary prostate cancer (CaP) and benign prostatic hyperplasia (BPH) cultures. To perform microvessel density (MVD) counts on sections of BPH and CaP from the same prostatectomy specimens. SCOPE: Angiogenic cytokine expression was measured by immunoassays and in vitro angiostatin generating capacities assessed using immunoblotting. CaP and BPH tissue was immunostained using factor VIII antibody to determine MVD. CONCLUSIONS: Elements regulating angiogenesis are present in both primary cultures of CaP and BPH, suggesting that angiogenic ability is well established in the absence of carcinoma.

Hydrotherapy during labor. An example of developing a practice policy.

Warm water has been used for centuries to treat many painful ailments. In perinatal clinical practice, hydrotherapy is being used to increase relaxation and decrease pain during labor. This article summarizes clinical studies evaluating hydrotherapy during labor, describing the current evidence which supports hydrotherapy as an effective method for managing intrapartal pain. The article also describes the process by which an institution established an evidence-based practice policy for the use of hydrotherapy in labor.

miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT.

Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3' untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model.

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a 'wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

Beyond prostate-specific antigen: alternate serum markers.

There is a need to improve existing methods for early diagnosis of prostate cancer (CaP) and to identify men at risk for developing aggressive disease. In an effort to replace and/or supplement prostate-specific antigen many serum analytes have been examined, but with little supportive data for clinical use. Recently, technological advances in molecular assays have improved investigational outcomes and have led to the discovery of a number of prospective markers with high specificity. Further promise for providing more accurate CaP diagnosis and prognosis lies in proteomic array profiling and DNA methylation assays. This review illustrates the current benefits and limitations of potentially useful CaP serum markers that have considerable existing data and touches upon other future markers as well as the field of proteomics.

Src family kinase oncogenic potential and pathways in prostate cancer as revealed by AZD0530.

Prostate cancer is the most frequently diagnosed cancer in American men. We have previously demonstrated that Src mediates androgen-independent proliferation in prostate cancer. We sought to investigate the Src-mediated oncogenic pathways and tumor biology using AZD0530, a novel Src family kinase/Abl dual-kinase inhibitor that is entering phase II clinical trials. We show that while both Src and Abl are expressed in all prostate cancer cell lines, Src but not Abl is activated in the prostate. Furthermore, Src activation is inhibited by AZD0530 in a rapid and dose-dependent manner. We show that Src mediates cell proliferation in DU145 and PC3 cells at the G1 phase of cell cycle. Src inhibition resulted in decreased binding of beta-catenin to the promoters of G1 phase cell cycle regulators cyclin D1 and c-Myc. C-Myc may also be regulated at the protein level by extracellular signal-regulated kinase 1/2 and GSK3beta. Cell motility factors focal adhesion kinase, p130CAS and paxillin activation in DU145 and PC3 cells were also inhibited. Administration of AZD0530 in mice reduced orthotopic DU145 xenograft growth by 45%. We have further delineated the Src-mediated oncogenic growth and migration pathways in prostate cancer and established mechanistic rationale for Src inhibition as novel therapy in the treatment of prostate cancer.

Inhibition of Akt pathways in the treatment of prostate cancer.

Akt is a serine/threonine kinase mediating multiple intracellular pathways involved in prostate cancer (CaP) biology. Increased understanding of the molecular mechanisms of Akt activation and signaling have led to the development of an increasing number of Akt inhibitors. These biologic agents demonstrate activity against a wide range of cancers in preclinical studies. Clinical studies of Akt inhibition in CaP are in progress, including agents such as celecoxib, perifosine and genistein. How best to integrate Akt inhibitors with standard CaP therapy or select patients most likely to benefit is the subject of ongoing research.

What to do if the lymph nodes are positive.

Lymph node metastases are common in patients with advanced bladder cancer. Survival rates have apparently improved from about 15% to 30% as management evolved from lymphadenectomy plus cystectomy to multimodal therapy, but investigators have not conclusively shown the benefit of adjuvant irradiation and/or chemotherapy. If clinical staging indicates nodal metastases, we recommend primary multiagent chemotherapy with subsequent exploratory laparotomy, lymphadenectomy, and possibly cystectomy for complete responders. Patients without clinical evidence of metastases undergo complete bilateral pelvic lymphadenectomy plus cystectomy if nodes are normal, or if grossly abnormal but resectable. Patients with nodal metastases may be candidates for adjuvant chemotherapy.