Layered Sheath-Assisted Dissection and Retrieval of Stuck Port Catheters: A Single-Center Experience.

Chemoports are often required for oncological patients requiring repeated blood draws and long-term drug therapy. However, complications such as dislodgement, fracture, thrombosis, and venous occlusion may occur if the ports remain unremoved when not in use. Nonetheless, existing techniques require multiple accesses or release of the stuck catheter tip to retrieve the catheter, making the procedure inconvenient. We present our experience with a technique using the Bard Denali inferior vena cava filter retrieval kit to remove a stuck or fractured chemoport catheter through a single vascular access. The technique was performed in two female patients with satisfactory results (complete retrieval of broken chemoports) and an event-free follow-up period. The entire procedure was completed within 15-30 minutes with fluoroscopic time under two minutes. The technique allows for better case management by simplifying the procedure, reducing radiation, and improving workflow efficiency in the operating room.

Thrombosis with Thrombocytopenia Syndrome (TTS) After ChAdOx1 nCoV-19 Immunization: An Investigative Case Report.

BACKGROUND Thrombosis with thrombocytopenia syndrome (TTS), including vaccine-induced immune thrombotic thrombocytopenia (VITT), is an extremely rare adverse effect, mostly seen after initial vaccination with the viral vector-based AstraZeneca-Oxford COVID-19 vaccine. It is characterized by mild to severe thrombocytopenia and venous or arterial thrombosis. CASE REPORT Herein, we present a case of an 18-year-old male patient who developed Level 1 TTS (probable VITT) eight days after immunization with the ChADOx1 nCOV-19 vaccine (Covishield; AZ-Oxford). Initial investigations revealed severe thrombocytopenia, hemiparesis, and intracranial hemorrhage, after which the patient was treated conservatively. However, a decompressive craniotomy was performed later due to patient deterioration. One week after surgery, the patient developed bilious vomiting, lower-gastrointestinal bleeding, and abdominal distension. An abdominal CT scan was performed that showed thrombosis of the portal vein with occlusion of the left iliac vein. The patient underwent an exploratory laparotomy followed by resection and anastomosis of the small bowel due to massive gut gangrene. Due to persistent thrombocytopenia after surgery, intravenous immune globulin (IVIG) was administered. The platelet count increased thereafter, and the patient stabilized. He was discharged on the 33rd day after admission and was followed up for a year. No post-hospitalization complications were observed in the follow-up period. CONCLUSIONS Although vaccines have been proven to be highly safe and effective to end the Coronavirus Disease 2019 (COVID-19) caused pandemic, there is still a small risk of developing rare complications, including TTS and VITT. Early diagnosis and prompt intervention are key for patient management.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.

miR-27b-5p inhibits BeWo cells fusion by regulating WNT2B and enzyme involved in progesterone synthesis.

PROBLEM: The miRNAs show placenta-specific expression patterns, which alter during pregnancy-related complications. In present study, the role of miR-27b-5p during forskolin-mediated BeWo cells fusion has been investigated. METHOD OF STUDY: The fusion of BeWo cells in response to forskolin treatment (25 µM) was studied by desmoplakin I+II staining. Expression profile of miR-27b-5p by qRT-PCR and its targets HSD3ß1 and WNT2B by qRT-PCR and in Western blot were studied. The effect of overexpression of miR-27b-5p and silencing of HSD3ß1 & WNT2B by siRNA on forskolin-mediated BeWo cells fusion and secretion of hCG and progesterone by ELISA was investigated. RESULTS: Time-dependent down-regulation in the expression of miR-27b-5p in forskolin-treated BeWo cells has been confirmed by qRT-PCR. Overexpression of miR-27b-5p significantly inhibits forskolin-mediated BeWo cells fusion as well as hCG & progesterone secretion. HSD3ß1 and WNT2B were identified as targets of miR-27b-5p and are up-regulated in forskolin-treated BeWo cells. Overexpression of miR-27b-5p in BeWo cells downregulates their expression. Further, luciferase reporter assay revealed that miR-27b-5p directly target expression of both HSD3ß1 and WNT2B. Silencing of both HSD3ß1 and WNT2B leads to a significant reduction in forskolin-mediated BeWo cells fusion with concomitant decrease in the secretion of progesterone or/and hCG. Decrease in forskolin-mediated cells fusion observed in miR-27b-5p mimic transfected BeWo cells could be rescued by the overexpression of both HSD3ß1 and WNT2B. CONCLUSION: These observations suggest that reduced miR-27b-5p in forskolin-treated BeWo cells leads to increased secretion of progesterone and hCG due to loss of repressional control on HSD3ß1 and WNT2B.

HGF regulate HTR-8/SVneo trophoblastic cells migration/invasion under hypoxic conditions through increased HIF-1α expression via MAPK and PI3K pathways.

Hepatocyte growth factor (HGF) is reported to be down-regulated in pregnancy complications like intrauterine growth retardation and preeclampsia, which are associated with abnormal trophoblast migration/invasion. In this study, role of HGF and associated signaling pathways has been investigated in HTR-8/SVneo trophoblastic cells migration/invasion under normoxia (20% O2) and hypoxia (2% O2). HTR-8/SVneo cells exposed to hypoxia showed increase in migration and invasion as compared to cells incubated under normoxic conditions. The migration/invasion under both normoxic and hypoxic conditions was further enhanced after treatment with HGF. Subsequent to treatment with HGF, a significant increase in expression of MMP2 & MMP3 under normoxia and MMP1 & MMP9 under hypoxia was observed. Treatment of HTR-8/SVneo cells with HGF under hypoxia also led to decrease in TIMP1. Treatment of the cells with HGF led to activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Inhibition of MAPK by U0126 and PI3K by LY294002 led to concomitant decrease in the HGF-mediated migration/invasion of HTR-8/SVneo cells. HGF treatment under hypoxia also led to a significant increase in hypoxia inducible factor (HIF-1α) expression. Additionally, inhibition of HIF-1α by siRNA led to decrease in HGF-mediated migration of HTR-8/SVneo cells under hypoxic conditions. Inhibition of HGF activated MAPK and PI3K signaling led to reduction in HIF-1α expression under hypoxia. In conclusion, HGF facilitates HTR-8/SVneo cell migration/invasion by activation of MAPK/PI3K signaling pathways and increased expression of MMPs. HIF-1α has a role in HGF-mediated increase in migration under hypoxic conditions.

HGF promotes HTR-8/SVneo cell migration through activation of MAPK/PKA signaling leading to up-regulation of WNT ligands and integrins that target ß-catenin.

Inadequate migration and invasion of the trophoblast cells during embryo implantation is one of the reasons for pregnancy-related complications such as intrauterine growth restriction and preeclampsia. In the present study, relevance of WNT ligands and integrins associated with hepatocyte growth factor (HGF)-mediated migration of HTR-8/SVneo trophoblastic cells has been investigated. Treatment of HTR-8/SVneo cells with HGF led to a dose-dependent increase in their migration. RT-PCR studies revealed a significant increase in the transcripts of WNT4, WNT11, ITGA2, and ITGAV, which was further confirmed at protein level by Western blotting. HGF treatment also led to increased expression of integrin α2ß1 and αVß5 in HTR-8/SVneo cells. Silencing of WNT4, WNT11, ITGA2, and ITGAV by siRNA led to a significant decrease in HGF-mediated migration of cells. Treatment of cells with HGF led to activation of mitogen-activated protein kinases (MAPK) and protein kinase A (PKA) signaling pathways. Inhibition of MAPK/PKA, by selective inhibitors, led to decrease in the expression of above WNT ligands and integrins. Silencing of WNT4/WNT11 led to concomitant decrease in the expression of ITGA2 and ITGAV and vice versa. HGF treatment also led to significant increase in ß-catenin expression, a downstream target of both WNT ligands and integrins. Silencing of ß-catenin led to decrease in HGF-mediated migration. ß-catenin expression was also down-regulated in WNT4/WNT11/ITGA2/ITGAV silenced cells suggesting a possible cross-communication of WNT ligands and integrins via ß-catenin. These studies have established the significance of WNT4/WNT11 as well as ITGA2/ITGAV during HGF-mediated migration of HTR-8/SVneo trophoblastic cells.

Relevance of Wnt10b and activation of ß-catenin/GCMa/syncytin-1 pathway in BeWo cell fusion.

PROBLEM: To study the involvement of specific Wnt(s) ligand during trophoblastic BeWo cell differentiation. METHOD OF STUDY: BeWo cells on treatment with forskolin/human chorionic gonadotropin (hCG) were studied for cell fusion by desmoplakin I+II staining and/or hCG secretion by ELISA. Levels of Wnt10b/ß-catenin/glial cell missing a (GCMa)/syncytin-1 were studied by qPCR/Western blotting in forskolin-/hCG-treated control siRNA and Wnt10b silenced BeWo cells. RESULTS: BeWo cells on treatment with hCG (5 IU/mL) led to a 94-fold increase in Wnt10b transcript. Wnt10b silencing showed significant decrease in forskolin-/hCG-mediated BeWo cell fusion and/or hCG secretion. It led to down-regulation of ß-catenin (nuclear and cytoplasmic), GCMa and syncytin-1 expression. Treatment of BeWo cells with H89, protein kinase A (PKA) signaling inhibitor, significantly reduced forskolin-/hCG-induced Wnt10b, ß-catenin, and syncytin-1 expression, which also resulted in reduced cell fusion. CONCLUSION: Wnt10b is involved in forskolin/hCG-mediated BeWo cell fusion via ß-catenin/GCMa/syncytin pathway, which may also involve activation of PKA.

Cell Signaling Pathways Involved During Invasion and Syncytialization of Trophoblast Cells.

Implantation involves an extensive cross talk between the trophoblast cells and the receptive endometrium through embryonic as well as endometrial-derived factors that regulate the invasion and migration of trophoblast cells and also syncytia formation. Any aberration in this highly regulated process may lead to pregnancy complications such as preeclampsia, intrauterine growth restriction, or even pregnancy failure. How various cytokines and growth factors act by activating various cell signaling pathways leading to the expression of the effector molecules have been reviewed, which control invasion and migration of trophoblast cells and syncytialization. The gaps in our current understanding of the various signaling pathways, activated by different cytokines/growth factors, their possible cross talk for optimized effector function(s), and future prospects in this field have been discussed.

Humanized antibody neutralizing 2009 pandemic H1N1 virus.

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (KD = 0.75 ± 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 µg/mL), with marginally higher IC50 as compared to MA2077 (0.08 µg/mL). In addition, 2077Hu2 also retained the epitope specificity for the "Sa" antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.