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International Journal of Molecular... May 2024Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells.... (Comparative Study)
Comparative Study
Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells. The aim of the study was to immortalize the primary ovarian cancer (OvCa) cells using the plasmid-carrying human telomerase reverse transcriptase (hTERT) gene and compare their phenotype and biological activity with the primary cells. The primary OvCa3 A and OvCa7 A cells were isolated from the ascitic fluid of two high-grade serous ovarian cancer patients and were characterized using immunocytochemical methods, flow cytometry, real-time RT-PCR, Western blot, metabolic activity, and migratory potential. Both immortalized ovarian cancer cell lines mirrored the phenotype of primary cancer cells, albeit with modifications. The OvCa3 A hTERT cells kept the mesenchymal stem cell phenotype of CD73/CD90/CD105-positivity and were CD133-negative, whereas the cell population of OvCa7 A hTERT lost CD73 expression, but almost 90% of cells expressed the CD133 characteristic for the CSCs phenotype. Immortalized OvCa cells differed in gene expression level with respect to and , which was associated with stemness properties. The OvCa7 A hTERT cells showed higher metabolic and migratory activity and ALDH1 expression than the corresponding primary OvCa cells. Both primary and immortalized cell lines were able to form spheroids. The newly established unique immortalized cell line OvCa7 A hTERT, with the characteristic of a serous ovarian cancer malignancy feature, and with the accumulation of the p53, Pax8, and overexpression of the CD133 and CD44 molecules, may be a useful tool for research on therapeutic approaches, especially those targeting CSCs in ovarian cancer and in preclinical 2D and 3D models.
Topics: Humans; Female; Ovarian Neoplasms; Telomerase; Cell Line, Tumor; Neoplastic Stem Cells; Cell Movement; Gene Expression Regulation, Neoplastic
PubMed: 38791431
DOI: 10.3390/ijms25105384 -
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... May 2024Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression...
Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:10. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.
Topics: Animals; Zika Virus; Mice, Inbred BALB C; Antibodies, Monoclonal; Viral Envelope Proteins; Mice; Humans; HEK293 Cells; Female; Antibodies, Viral; Protein Domains; Enzyme-Linked Immunosorbent Assay
PubMed: 38790101
DOI: No ID Found -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the...
The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.
Topics: tau Proteins; Animals; Antibodies, Monoclonal; Humans; Mice; Alzheimer Disease; Enzyme-Linked Immunosorbent Assay; Recombinant Proteins; Hybridomas; Mice, Inbred BALB C; Antibody Specificity; Protein Domains; Epitopes
PubMed: 38783817
DOI: 10.13345/j.cjb.230655 -
Journal of Cytology 2024The "International System of Reporting Serous Fluid Cytology (TIS)" together with cytomorphology promotes the use of ancillary techniques to resolve difficulties in...
BACKGROUND
The "International System of Reporting Serous Fluid Cytology (TIS)" together with cytomorphology promotes the use of ancillary techniques to resolve difficulties in reporting serous fluid cytology.
OBJECTIVE
To classify serous effusion fluid samples received at our department in line with "TIS", indicating the risk of malignancy (ROM), and directing appropriate usage of ancillary testing.
MATERIALS AND METHODS
Prospective study carried out from October 2021 to September 2022. The study included all pleural, ascitic, and pericardial fluid samples, reported according to 'TIS'. Flow cytometry and immunocytochemistry were ancillary methods utilized to assist in reporting. Cases with available history and convincing correlations didn't require further assessment.
RESULTS
A total of 1200 serous effusion samples were evaluated including 604 pleural, 591 ascitic, and 5 pericardial fluid samples. After categorization, there were 23 samples in non-diagnostic (ND, 1.9%), 575 in negative for malignancy (NFM, 47.91%), 44 in atypia of undetermined significance (AUS, 3.66%), 64 in suspicious for malignancy (SFM, 5.33%), and 494 in malignant category (MAL, 41.16%). Ancillary studies were beneficial in the recategorization of 26% (11/44) AUS cases, 29.6% (19/64) SFM cases, and it helped refine tumor characteristics in 35.42% (175/494) cases categorized as malignant. Final ROM calculated for each category: ND 25%, NFM 18.6%, AUS 66.6%, SFM 88%, and MAL 100%.
CONCLUSION
Serous fluid is an easily obtainable sample that can provide opportunities for ancillary testing with clinical implications. In AUS and suspicious category although, diagnostic yield is increased however, a larger number of cases are required to obtain definite results.
PubMed: 38779601
DOI: 10.4103/joc.joc_114_23 -
Antimicrobial Stewardship & Healthcare... 2024Assess whether direct inoculation of ascites into blood culture bottles would improve ascites culture yield.
OBJECTIVE
Assess whether direct inoculation of ascites into blood culture bottles would improve ascites culture yield.
DESIGN
Pre-post-study.
SETTING
The study was performed at a quaternary academic medical center in Houston, Texas, including all inpatient and emergency department encounters.
PATIENTS
Ascites cultures collected from November 2020 to December 2022 were reviewed and screened for spontaneous bacterial peritonitis. Patients were excluded if a prior ascites culture from the same patient was already included in the study or if there was evidence of secondary bacterial peritonitis.
INTERVENTION
In the pre-intervention period, ascites cultures were collected into a sterile container and inoculated onto/into solid and liquid media. In the post-intervention period, ascites cultures were instead directly inoculated into bioMérieux© blood culture bottles at the bedside.
RESULTS
114 patients met inclusion and exclusion criteria, 61 pre-intervention and 53 post-intervention. Overall ascites culture positivity was 15.8% (18/114), 11.5% (7/61) pre-intervention vs 20.8% (11/53) post-intervention. After adjusting for confounders, the intervention had a trend toward a significant effect on ascites culture positivity ( = 0.077). No significant differences were seen in time to positivity, hospital length of stay, or 30-day readmission.
CONCLUSIONS
Direct inoculation of ascitic fluid into blood culture bottles led to a small increase in culture yield but lacked statistical significance. This lack of significance may be due to the study being underpowered. Further studies are required to investigate if this is due to procedural inefficiencies (eg, inadequate inoculation volumes) or pragmatic clinical practice considerations (ie, high rates of pre-culture antibiotics).
PubMed: 38774119
DOI: 10.1017/ash.2024.84 -
BMC Women's Health May 2024Concomitant invasive ovarian mucinous adenocarcinoma, unilateral renal agenesis and bicornuate uterus is a rare combination. Unilateral renal agenesis has been...
BACKGROUND
Concomitant invasive ovarian mucinous adenocarcinoma, unilateral renal agenesis and bicornuate uterus is a rare combination. Unilateral renal agenesis has been associated with genital anomalies, such as unicornuate and bicornuate uterus. Furthermore, a wealth of studies has reported the association between unicornuate uterus and ovarian anomalies, such as the absence of an ovary or ectopic ovaries, but rarely has there been a combination of the three to the best of our knowledge. The present case report is the first case presentation with a combination of the three syndromes: ovarian mucinous tumor, unilateral renal agenesis, and bicornuate uterus.
CASE PRESENTATION
We report the case of a 17-year-old who presented with abdominal distension. On examination, a CT scan revealed a large multicystic abdominal mass on the right side, with an absence of the right kidney while the left kidney was normal in size, appearance, and position. Intraoperatively, massive blood-stained ascitic fluid was evacuated. Additionally, a large whitish polycystic intra-abdominal mass with mucus-like materials and solid areas was attached to the midpoint of the colon and the right ovary, with visible metastasis to the omentum. The uterus was bicornuate. The mass and omentum were taken for histopathology and a diagnosis of invasive ovarian mucinous cystadenocarcinoma with metastasis to the colon and omentum was made after a pathological report.
CONCLUSIONS
The presence of these conditions in the same individual could potentially complicate medical management and fertility considerations. Thus, a need for a multidisciplinary medical team, including gynecologists, urologists, and oncologists, to address their unique needs and provide appropriate treatment and guidance. Further research and case studies are needed to better understand the possible association and implications of these rare co-occurring conditions.
Topics: Adolescent; Female; Humans; Adenocarcinoma, Mucinous; Bicornuate Uterus; Congenital Abnormalities; Kidney; Ovarian Neoplasms; Solitary Kidney; Tomography, X-Ray Computed; Uterus
PubMed: 38769573
DOI: 10.1186/s12905-024-03130-y -
BMC Infectious Diseases May 2024Metagenomic next-generation sequencing (mNGS) is an emerging technique for the clinical diagnosis of infectious disease that has rarely been used for the diagnosis of...
BACKGROUND
Metagenomic next-generation sequencing (mNGS) is an emerging technique for the clinical diagnosis of infectious disease that has rarely been used for the diagnosis of ascites infection in patients with cirrhosis. This study compared mNGS detection with conventional culture methods for the on etiological diagnosis of cirrhotic ascites and evaluated the clinical effect of mNGS.
METHODS
A total of 109 patients with ascites due to cirrhosis were included in the study. We compared mNGS with conventional culture detection by analyzing the diagnostic results, pathogen species and clinical effects. The influence of mNGS on the diagnosis and management of ascites infection in patients with cirrhosis was also evaluated.
RESULTS
Ascites cases were classified into three types: spontaneous bacterial peritonitis (SBP) (16/109, 14.7%), bacterascites (21/109, 19.3%) and sterile ascites (72/109, 66.1%). In addition, 109 patients were assigned to the ascites mNGS-positive group (80/109, 73.4%) or ascites mNGS-negative group (29/109, 26.6%). The percentage of positive mNGS results was significantly greater than that of traditional methods (73.4% vs. 28.4%, P < 0.001). mNGS detected 43 strains of bacteria, 9 strains of fungi and 8 strains of viruses. Fourteen bacterial strains and 3 fungal strains were detected via culture methods. Mycobacteria, viruses, and pneumocystis were detected only by the mNGS method. The mNGS assay produced a greater polymicrobial infection rate than the culture method (55% vs. 16%). Considering the polymorphonuclear neutrophil (PMN) counts, the overall percentage of pathogens detected by the two methods was comparable, with 87.5% (14/16) in the PMN ≥ 250/mm group and 72.0% (67/93) in the PMN < 250/mm group (P > 0.05). Based on the ascites PMN counts combined with the mNGS assay, 72 patients (66.1%) were diagnosed with ascitic fluid infection (AFI) (including SBP and bacterascites), whereas based on the ascites PMN counts combined with the culture assay, 37 patients (33.9%) were diagnosed with AFI (P < 0.05). In 60 (55.0%) patients, the mNGS assay produced positive clinical effects; 40 (85.7%) patients had their treatment regimen adjusted, and 48 patients were improved. The coincidence rate of the mNGS results and clinical findings was 75.0% (60/80).
CONCLUSIONS
Compared with conventional culture methods, mNGS can improve the detection rate of ascites pathogens, including bacteria, viruses, and fungi, and has significant advantages in the diagnosis of rare pathogens and pathogens that are difficult to culture; moreover, mNGS may be an effective method for improving the diagnosis of ascites infection in patients with cirrhosis, guiding early antibiotic therapy, and for reducing complications related to abdominal infection. In addition, explaining mNGS results will be challenging, especially for guiding the treatment of infectious diseases.
Topics: Humans; Liver Cirrhosis; Male; High-Throughput Nucleotide Sequencing; Female; Middle Aged; Ascites; Metagenomics; Peritonitis; Aged; Bacterial Infections; Adult; Bacteria; Ascitic Fluid
PubMed: 38769522
DOI: 10.1186/s12879-024-09396-9 -
Revista Brasileira de Ginecologia E... 2024
Topics: Humans; COVID-19; Ascitic Fluid; SARS-CoV-2
PubMed: 38765511
DOI: 10.61622/rbgo/2024rbgo24 -
Surgery Today Apr 2024To compare the pathophysiology and surgical outcomes of emergency surgery for upper gastrointestinal tract perforation with and without fungal peritonitis and identify...
PURPOSE
To compare the pathophysiology and surgical outcomes of emergency surgery for upper gastrointestinal tract perforation with and without fungal peritonitis and identify the risk factors for fungal peritonitis.
METHODS
The subjects of this retrospective study were patients with upper gastrointestinal perforation and peritonitis who underwent emergency surgery at a single medical center in Japan. The patients were allocated to two groups according to the presence or absence of fungal peritonitis: group F and group N, respectively.
RESULTS
At the time of surgery, ascitic fluid culture or serum β-D glucan levels were available for 54 patients: 29 from group F and 25 from group N, respectively. The stomach was perforated in 14 patients (25.9%) and the duodenum was perforated in 40 patients (74.1%). Group F had a higher proportion of patients with low preoperative prognostic nutritional index scores (≤ 40) and C-reactive protein levels and a higher postoperative complication rate. The time to initiate food intake and the postoperative hospital stay were also significantly longer in group F. Multivariate analysis identified that the perforation site of the stomach was a risk factor for fungal peritonitis.
CONCLUSION
Patients with fungal peritonitis from upper gastrointestinal tract perforation had higher postoperative complication rates, delayed postoperative recovery, and a longer hospital stay. Gastric perforation was a risk factor for fungal peritonitis.
PubMed: 38691220
DOI: 10.1007/s00595-024-02851-9 -
Veterinary Clinical Pathology Jun 2024A 9-year-old dog was presented with weight loss, respiratory effort, and an enlarged abdomen. Imaging studies and exploratory surgery showed pulmonary and splenic masses...
A 9-year-old dog was presented with weight loss, respiratory effort, and an enlarged abdomen. Imaging studies and exploratory surgery showed pulmonary and splenic masses and bi-cavitary effusion, later classified as hemorrhage. Cytology of the peritoneal and pleural fluids also revealed several microfilariae. Immunologic and molecular analyses confirmed Dirofilaria immitis infection and histopathology of the spleen indicated a cavernous endothelial proliferation with undefined etiology (hemangiosarcoma vs reaction to parasite infestation). The nematode larvae are speculated to have entered body cavities via erratic migration or via hemorrhage and visceral lesions to be related to parasitism. Nematode infection should be considered as a differential diagnosis for internal bleeding of undetermined origin.
Topics: Animals; Dogs; Dirofilariasis; Dog Diseases; Dirofilaria immitis; Hemorrhage; Male; Spleen; Ascitic Fluid
PubMed: 38684482
DOI: 10.1111/vcp.13351