Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.

Disconnected Pancreatic Duct Syndrome: A Rare Complication of Pancreatitis.

Disconnected pancreatic duct syndrome (DPDS) is a rare complication of a common disease. Typically, DPDS occurs in acute necrotizing pancreatitis (ANP), chronic pancreatitis, abdominal surgery, or trauma. We present a case of DPDS from acute non-necrotizing pancreatitis (ANNP). A 41-year-old male with a history of alcohol use and prior AP presented with progressive, severe left-sided abdominal pain that was worse with movement. Labs revealed a lipase of 95 U/L (normal range 11-82 U/L). Computed tomography (CT) of the abdomen/pelvis (A/P) with IV contrast demonstrated a large left-sided pleural effusion, non-necrotic pancreatic pseudocysts, and a large subdiaphragmatic fluid collection. Thoracentesis of the pleural effusion revealed an amylase of 601 U/L confirming pancreatic etiology. A subsequent magnetic resonance cholangiopancreatography (MRCP) confirmed complex peripancreatic ascites, rapid subdiaphragmatic fluid accumulation, and a fistula from the pancreatic tail to retroperitoneum concerning for a rapidly dissecting pancreatic pseudocyst. He ultimately underwent endoscopic retrograde cholangiopancreatography (ERCP) with stent placement in the main pancreatic duct. His left-sided abdominal pain rapidly improved, and the patient was discharged. CT A/P one week after discharge showed a reduced size of subdiaphragmatic fluid collection. DPDS is usually seen in patients with a history of ANP. Our case demonstrates that it can also occur in ANNP, which has not previously been described in the literature. Therefore, a high index of clinical suspicion must be maintained for DPDS even in ANNP given its potential for severe complications.

Association of preoperative workup and comorbidities with risk of gastroesophageal surgery failure.

Background: While surgical failure rates for fundoplication and hiatal hernia repair are low, there has been no clear evaluation of the preoperative risk factors associated with surgical failure. This study aimed to identify risk factors predisposing patients to surgical failure. Methods: Patients who underwent antireflux surgery during a 3-year period were evaluated for evidence of surgical complications and placed accordingly into the failure or control group. Demographic data, comorbidities, clinical presentation, preoperative evaluation, and surgical data were collected and compared between the groups. Results: In total, 86 patients with failure and 42 controls were identified among our cohort. No significant differences were found between groups based on sex (P=0.640). However, patients with failure were younger than controls (57.0 vs. 64.7 years, P=0.0001). Body mass index, tobacco use and alcohol use did not differ significantly between the groups (P=0.189, P=0.0999, P=0.060). Notably, psychiatric illness was more common in the failure group (P=0.0086). Neither hypertension (P=0.134) nor diabetes (P=0.335) had significant differences between groups. For procedures, no significant differences were found for the frequencies of preoperative imaging (P=0.395) or manometry (P=0.374), but pH/BRAVO studies (P=0.0193) and endoscopy (P<0.001) were both performed more frequently in the failure group. Conclusions: Patients with psychiatric comorbidities are at higher risk of surgical failure. Alcohol use trended toward significance, which warrants further investigation. We also noted an increase in rates of preoperative pH and endoscopy studies, contrary to the prior literature; this is likely due to more complex cases requiring additional workup.

A Curious Case of Rectal Ejaculation.

Rectal-prostate fistulas are uncommon anatomical connections between the prostatic urethra and rectum that are typically iatrogenic but can also result from other underlying pathology. Here, we present a unique case of a rectal-prostate fistula causing the rectal passage of sperm. A 33-year-old male with a history of illicit drug use presented with five days of testicular pain and a substantial amount of sperm passage from his rectum with ejaculation for the past two years. Computed tomography and voiding cystourethrogram (VCUG) of the pelvis revealed evidence of a rectal-prostate fistula. He was treated with piperacillin-tazobactam, and a surgical fistula repair was performed. Further investigation divulged a three-week comatose state due to cocaine and phencyclidine intoxication two years prior with documentation suggesting a traumatic Foley catheter placement and strong suspicion for premature balloon dilation in the prostatic urethra. Repeat VCUG revealed resolution of the fistula with mildly reduced antegrade ejaculatory volume. Cases secondary to Foley catheter placement have not been previously reported in the literature. Even though urethral catheters have been shown to be effective tools in healthcare, it is crucial for clinicians to recognize the numerous potential complications that oftentimes become an afterthought to many providers. This case not only highlights a rare complication of catheter use but also emphasizes the importance of provider mindfulness when utilizing seemingly benign therapies such as Foley catheters.

Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer's disease, progressive supranuclear palsy and dementia with Lewy bodies.

Tau aggregates propagate in brain cells and transmit to neighboring cells as well as anatomically connected brain regions by prion-like mechanisms. Soluble tau aggregates (tau oligomers) are the most toxic species that initiate neurodegeneration in tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Exogenous tau aggregates have been shown to be internalized by brain cells; however, the precise cellular and molecular mechanisms that underlie the internalization of tau oligomers (TauO) remain elusive. Using brain-derived tau oligomers (BDTOs) from AD, PSP, and DLB patients, we investigated neuronal internalization mechanisms of BDTOs, including the heparan sulfate proteoglycan (HSPG)-mediated pathway, clathrin-mediated pathway, and caveolae-mediated pathway. Here, we demonstrated that the HSPG-mediated pathway regulates internalization of BDTOs from AD and DLB, while HSPG-mediated and other alternative pathways are involved in the internalization of PSP-derived tau oligomers. HSPG antagonism significantly reduced the internalization of TauO, prevented tau translocation to the endosomal-lysosomal system, and decreased levels of hyperphosphorylated tau in neurons, the well-known contributor for neurofibrillary tangles (NFT) accumulation, degeneration of neurons, and cognitive decline. Furthermore, siRNA-mediated silencing of heparan sulfate (HS)-synthesizing enzyme, exostosin-2, leads to decreased internalization of BDTOs, prevented tau-induced autophagy-lysosomal pathway impairment, and decreased hyperphosphorylated tau levels. Collectively, these findings suggest that HSPG-mediated endocytosis and exostsin-2 are involved in neuronal internalization of TauO and subsequent tau-dependent neuropathology in AD and DLB.