Upper gastrointestinal endoscopy in the surgically altered patient.

As management of upper gastrointestinal malignancies improves, and with popularization of bariatric surgery, endoscopists are likely to meet patients with altered upper gastrointestinal anatomy. Short-term, the surgery can cause complications like bleeding, leaks, and fistulas, and longer-term problems such as intestinal or biliary anastomotic strictures or biliary stones can arise, all necessitating endoscopy. In addition, the usual upper gastrointestinal pathologies can also still occur. These patients pose unique challenges. To proceed, understanding the new layout of the upper gastrointestinal tract is essential. The endoscopist, armed with a clear plan for navigation, can readily diagnose and manage most commonly occurring conditions, such as marginal ulcers and proximal anastomotic strictures with standard endoscopic instruments. With complex reconstructions involving long segments of small bowel, such as Roux-en-Y gastric bypass, utilization of balloon-assisted enteroscopy may be necessary, mandating modification of procedures such as endoscopic retrograde cholangiopancreatography. Successful endoscopic management of patients with altered anatomy will require prior planning and preparation to ensure the appropriate equipment, setting, and skill set is provided.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps to chromosome 2 (locus 2q31.1), near Titin (TTN), which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in failing human heart tissue (HF) compared to non-failing hearts (NF). Using the Sanger sequencing method, we characterized the human OLA1 gene and screened for mutations in the OLA1 gene in patients with failing and non-failing hearts. Among failing and non-failing heart patients, we found 15 different mutations in the OLA1 gene, including two transversions, one substitution, one deletion, and eleven transitions. All mutations were intronic except for a non-synonymous 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results demonstrate that this PCR test can effectively screen for OLA1 mutation-associated cardiomyopathy in human patients using easily accessible cells or tissues, such as blood cells. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

A novel point-of-care test for cirrhosis based on dimeric to monomeric IgA ratio in blood: a pilot cohort study.

BACKGROUND AND AIMS: Dimeric IgA to monomeric IgA ratio (dIgA ratio) is a biomarker of gut mucosal leakage in liver cirrhosis. We evaluated the diagnostic performance of a novel point-of-care (POC) dIgA ratio test for cirrhosis. METHODS: Plasma samples from people with chronic liver disease were analyzed using the BioPoint POC dIgA ratio antigen immunoassay lateral flow test. Cirrhosis was defined by Fibroscan>12.5 kPa, clinical evidence of cirrhosis or liver histopathology. POC dIgA test diagnostic accuracy was determined in a test cohort using receiver operating characteristic curve analysis; optimal cutoffs for sensitivity and specificity were then applied to a validation cohort. RESULTS: A total of 1478 plasma samples from 866 patients with chronic liver disease were included (test cohort n = 260, validation cohort n = 606). In all, 32% had cirrhosis; 44% Child-Pugh A, 26% Child-Pugh B, and 29% Child-Pugh C. Median POC dIgA ratio was higher in cirrhosis (0.9) compared with no cirrhosis (0.4, p < 0.001), and in Child-Pugh class B/C compared with A cirrhosis (1.4 Child-Pugh B/C vs. 0.6 Child-Pugh A, p < 0.001). POC dIgA ratio test had good diagnostic accuracy for liver cirrhosis in the test cohort (area under the receiver operating characteristic curve=0.80); a dIgA ratio cutoff of 0.6 had a sensitivity of 74% and specificity of 86%. POC dIgA test accuracy was moderate in the validation cohort (area under the receiver operating characteristic curve=0.75; positive predictive value 64%, negative predictive value 83%). Using a dual cutoff approach, 79% of cirrhosis cases were correctly diagnosed and further testing was avoided in 57%. CONCLUSIONS: POC dIgA ratio test had moderate accuracy for cirrhosis. Further studies evaluating the accuracy of POC dIgA ratio testing for cirrhosis screening are warranted.

Validation of a novel point-of-care test for alanine aminotransferase measurement: A pilot cohort study.

BACKGROUND: Alanine aminotransferase (ALT) measurement is essential for evaluation of liver disease. We validated a novel rapid point-of-care (POC) test for ALT1 against laboratory ALT. METHODS: Stored plasma samples from adults with chronic liver disease (Test cohort n = 240; Validation cohort n = 491) were analysed using the BioPoint® antigen immunoassay POC ALT1 lateral flow test, which provides quantitative ALT results (Axxin handheld reader) or semi-quantitative results (visual read, cut off 40 IU/ml). The accuracy of POC ALT1 to detect ALT > 40 IU/L was determined by ROC analysis. In patients with chronic hepatitis B, treatment eligibility (EASL criteria) was determined using POC ALT1 and compared to laboratory ALT. RESULTS: POC ALT1 test had good accuracy for laboratory ALT > 40 IU/L: AUROC 0.93 (95% CI: 0.89-0.96) in the Test cohort and AUROC 0.92 (95% CI: 0.88-0.95) in the Validation cohort. POC ALT1 cut off of 0.8 for ALT > 40 IU/L maximised sensitivity (97%) and specificity (71%) in the Test cohort (42% laboratory ALT > 40 IU/L) and yielded PPV 84% and NPV 91% in the Validation cohort (19% laboratory ALT > 40 IU/L). Semi-quantitative POC ALT1 had good accuracy for laboratory ALT in the Validation cohort (AUROC 0.85, 95% CI: 0.81-0.99; sensitivity 77% and specificity 93%). Combined with HBV DNA and transient elastography, both quantitative and semi-quantitative POC ALT1 tests had good accuracy for excluding hepatitis B treatment needs (sensitivity 96%, specificity 78% and NPV 99%). CONCLUSION: The POC ALT1 test had good accuracy for elevated ALT levels and for determining treatment eligibility among people with chronic hepatitis B.

Autologous T cell responses to primary human colorectal cancer spheroids are enhanced by ectonucleotidase inhibition.

OBJECTIVE: T cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type. DESIGN: In a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response. RESULTS: We show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures. CONCLUSION: In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

A Confocal Microscopic Study on Percentage Penetration of Different Sealers into Dentin.

CONTEXT: Three-dimensional seal of the root canal space has always been challenging. The incorporation of gutta-percha and sealer prevents microleakage by bacteria, ensuring a shielded root canal space. However, the penetration of sealer to various depths within the root canal has always been looked with curiosity. AIMS: Thus, the present in vitro study was undertaken to evaluate the percentage and average depth of penetration of Endoflas F. S., AH Plus, and Epiphany sealers into dentinal tubules among the coronal, middle, and apical thirds of the roots following obturation with a lateral compaction technique using Confocal Laser Scanning Microscopy. SETTINGS AND DESIGN: The study is an in vitro randomized control trial. SUBJECTS AND METHODS: Thirty sound central incisors were decoronated at the level of the cementoenamel junction. Working length determination was done followed by a meticulous cleaning, shaping, and under copious irrigation. The teeth were then randomly divided into three groups: Endoflas FS sealer, AH Plus sealer, and Epiphany sealer. On fluorescence treatment, the teeth were sectioned at the midpoint of coronal, middle, and apical third of each root and viewed under confocal laser scanning microscope. STATISTICAL ANALYSIS USED: The results were analyzed using one-way ANOVA, and the significant difference between groups was analyzed with post hoc Tukey test. RESULTS: Epiphany sealer provided with better percentage and depth penetration in comparison to Endoflas FS and AH Plus sealers. Furthermore, the coronal third of the root had better percentage and sealer penetration than the middle and apical thirds. CONCLUSIONS: Sealers tend to provide a firm bond between the tooth and the gutta-percha. They bind, lubricate, and seal the gutta-percha cones to fill the accessory canals. Within the limitations of this study, the superior flow and enhanced setting time Epihany sealers provide better percentage and depth of penetration than AH Plus and Endoflas FS.

Dimeric IgA is a specific biomarker of recent SARS-CoV-2 infection.

Current tests for SARS-CoV-2 antibodies (IgG, IgM, IgA) cannot differentiate recent and past infections. We describe a point of care, lateral flow assay for SARS-CoV-2 dIgA based on the highly selective binding of dIgA to a chimeric form of secretory component (CSC), that distinguishes dIgA from monomeric IgA. Detection of specific dIgA uses a complex of biotinylated SARS-CoV-2 receptor binding domain and streptavidin-colloidal gold. SARS-CoV-2-specific dIgA was measured both in 112 cross-sectional samples and a longitudinal panel of 362 plasma samples from 45 patients with PCR-confirmed SARS-CoV-2 infection, and 193 discrete pre-COVID-19 or PCR-negative patient samples. The assay demonstrated 100% sensitivity from 11 days post-symptom onset, and a specificity of 98.2%. With an estimated half-life of 6.3 days, dIgA provides a unique biomarker for the detection of recent SARS-CoV-2 infections with potential to enhance diagnosis and management of COVID-19 at point-of-care.

Erratum: Resuming endoscopy during COVID-19 pandemic: ESGE, WEO and WGO Joint Cascade Guideline for Resource Limited Settings.

[This corrects the article DOI: 10.1055/a-1400-9135.].

Role of Endoscopy in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a progressive disease of the bile ducts that usually results in chronic liver disease often requiring liver transplantation. Endoscopy remains crucial to the care of these patients, although magnetic resonance cholangiopancreatography has replaced endoscopic retrograde cholangiopancreatography (ERCP) as the primary imaging modality for diagnosis. For detection of dysplasia or cholangiocarcinoma, ERCP with intraductal sampling remains compulsory. Moreover, dominant strictures play an important part in the disease development, and management by balloon dilatation or stenting could contribute to long-term prognosis. In addition, endoscopy offers management for adverse events such as bile leaks and anastomotic strictures after liver transplantation. Finally, the special phenotype of inflammatory bowel disease associated with PSC as well as the frequent occurrence of portal hypertension mandates close follow-up with colonoscopy and upper endoscopy. With the emergence of novel techniques, the endoscopist remains a key member of the multidisciplinary team caring for PSC patients.

Impact of the COVID-19 pandemic on gastrointestinal endoscopy in Africa.

Background and study aims As with all other fields of medical practice, gastrointestinal endoscopy has been impacted by the COVID-19 pandemic. However, data on the impact of the pandemic in Africa, especially sub-Saharan Africa are lacking. Methods A web-based survey was conducted by the International Working Group of the European Society for Gastrointestinal Endoscopy and the World Endoscopy Organization to determine the impact and effects the COVID-19 pandemic has had on endoscopists in African countries. Results Thirty-one gastroenterologists from 14 countries in north, central, and sub-Saharan Africa responded to the survey. The majority of respondents reduced their endoscopy volume considerably. Personal protective equipment including FFP-2 masks were available in almost all participating centers. Pre-endoscopy screening was performed as well. Conclusion The COVID-19 pandemic has had a substantial impact on gastrointestinal endoscopy in most African countries; however, the impact may not have been as devastating as expected.

A Region of UNC-89 (Obscurin) Lying between Two Protein Kinase Domains Is a Highly Elastic Spring Required for Proper Sarcomere Organization.

In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.

Dysbiosis of the Duodenal Mucosal Microbiota Is Associated With Increased Small Intestinal Permeability in Chronic Liver Disease.

OBJECTIVES: Chronic liver disease (CLD) is associated with both alterations of the stool microbiota and increased small intestinal permeability. However, little is known about the role of the small intestinal mucosa-associated microbiota (MAM) in CLD. The aim of this study was to evaluate the relationship between the duodenal MAM and both small intestinal permeability and liver disease severity in CLD. METHODS: Subjects with CLD and a disease-free control group undergoing routine endoscopy underwent duodenal biopsy to assess duodenal MAM by 16S rRNA gene sequencing. Small intestinal permeability was assessed by a dual sugar (lactulose: rhamnose) assay. Other assessments included transient elastography, endotoxemia, serum markers of hepatic inflammation, dietary intake, and anthropometric measurements. RESULTS: Forty-six subjects (35 with CLD and 11 controls) were assessed. In subjects with CLD, the composition (P = 0.02) and diversity (P < 0.01) of the duodenal MAM differed to controls. Constrained multivariate analysis and linear discriminate effect size showed this was due to Streptococcus-affiliated lineages. Small intestinal permeability was significantly higher in CLD subjects compared to controls. In CLD, there were inverse correlations between microbial diversity and both increased small intestinal permeability (r = -0.41, P = 0.02) and serum alanine aminotransferase (r = -0.35, P = 0.04). Hepatic stiffness was not associated with the MAM. DISCUSSION: In CLD, there is dysbiosis of the duodenal MAM and an inverse correlation between microbial diversity and small intestinal permeability. TRANSLATIONAL IMPACT: Strategies to ameliorate duodenal MAM dysbiosis may ameliorate intestinal barrier dysfunction and liver injury in CLD.

Human papillomavirus E7 oncoprotein expression by keratinocytes alters the cytotoxic mechanisms used by CD8 T cells.

Cervical cancer is a malignant transformation of keratinocytes initiated by the E7 oncoprotein of human papillomavirus (HPV). These tumors are characterized by keratinocyte hyperproliferation and are often infiltrated with activated CD8 T cells. HPV infection confers changes to gain immunological advantage to promote chronic infection, and these persist with malignant transformation. We investigated the relative importance of the many redundant mechanisms of cytotoxicity used by CD8 T cells to kill keratinocytes expressing HPV E7 oncoprotein using extended-duration time-lapse microscopy that allows examination of cell-to-cell interactions during killing. E7 expression by keratinocytes increased susceptibility to cell-mediated killing. However, while killing of non-transgenic keratinocytes was traditional, perforin-mediated, and caspase-dependent, E7-expression favored killing by perforin-independent, caspase-independent mechanisms. The roles of perforin, TNFα, IFNγ, Fas/FasL and PD1/PD-L1 were graded according to target cell survival to produce a hierarchy of killing mechanisms utilized in killing E7-expressing cells. TNFα was essential for perforin-mediated killing of E7-expressing cells, but not perforin-independent killing. IFNγ facilitated killing by Fas/FasL interaction, especially in the absence of perforin. Additionally, expression of E7 offered protection from killing by up regulation of PD-L1, Fas and FasL expression on keratinocytes promoting fight-back by target cells, resulting in effector cell death. This study shows that keratinocytes expressing E7 are highly susceptible to killing by CD8 T cells, but utilizing different armamentarium. Down-regulation of CD8 T cell cytotoxicity in HPV-related tumors may be due to suppression by E7-expressing keratinocytes. Immunotherapy for HPV-related cancers may be improved by suppression of PD-L1, or by suppression of FasL.

Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity.

Interferon gamma (IFNγ) is a key moderator of cell-mediated immunity with diverse, mainly pro-inflammatory actions on immunocytes and target tissue. Recent studies have shown it may enhance anti-tumor and antiviral effects of CD8 T cells. Here we investigate the mechanisms by which IFNγ mediates CD8 T-cell cytotoxic function. We show that in vivo, antigen-specific CD8 T cells that produce INFγ are necessary to effect rejection of skin grafts expressing OVA as a transgene in keratinocytes. The ability of CD8 T cells to produce IFNγ enhanced their ability to migrate to the site of antigen-presenting skin cells. By in vivo imaging, we show that CTL motility, particularly speed, during graft rejection was enhanced by locally available IFNγ. We then used a reductionist two-cell model of CTL effectors and keratinocyte targets to investigate the effects of locally available (paracrine) and CTL-producing (autocrine) IFNγ on the motility behavior and killing ability of the CTL. Using live-cell imaging by prolonged time-lapse microscopy of primary effector CD8 T cells and antigen-expressing primary keratinocyte targets, we show that CD8 T-cell cytotoxic function and motility is enhanced by locally available IFNγ. Conversely, deprivation of either autocrine or paracrine IFNγ, or blockade of IFNγ signaling to CTL markedly reduced their cytotoxic function, their kinematics, and effector cell survival. We conclude that in vitro and in vivo, autocrine production of IFNγ by CTL enhances their motility and promotes killing of primary target keratinocytes. The absolute need for local IFNγ to enable cytotoxic CD8 T-cell function is of significance for immunotherapy for chronic viral infection and for cancer.