Multiple antibody detection in 'seronegative' myasthenia gravis patients.

BACKGROUND AND PURPOSE: Myasthenia gravis (MG) is an autoimmune disease caused by antibody mediated impairment in the neuromuscular junction. Seronegative MG (SNMG) without antibodies against acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) by routine assays accounts for about 20% of all MG patients. METHODS: Plasma from 81 Chinese MG patients previously found to be seronegative was tested by routine assays for AChR and MuSK antibodies. These samples were screened by (i) a novel, highly sensitive radioimmunoassay for AChR antibodies; (ii) cell-based assays for clustered AChR, MuSK and lipoprotein receptor-related protein 4 (LRP4) antibodies; (iii) a radioimmunoassay for titin antibodies. RESULTS: Antibodies to AChR, MuSK, LRP4 and titin were found in 25% (20/81), 4% (3/81), 7% (6/81) and 6% (5/78) of SNMG patients, respectively. In total, 37% of SNMG patients were found to be positive for at least one of the tested antibodies. AChR antibody positive patients had more severe disease (P = 0.008) and a trend towards fewer remissions/minimal manifestations than AChR antibody negative patients. The four patients with coexistence of antibodies had more severe disease, whilst the seronegative patients had milder MG (P = 0.015). CONCLUSIONS: Detection of multiple muscle antibodies by more sensitive assays provides additional information in diagnosing and subgrouping of MG and may guide MG treatment.

The prognostic utility of serum thyrotropin in hospitalized Covid-19 patients: statistical and machine learning approaches.

PURPOSE: To assess the prognostic value of serum TSH in Greek patients with COVID-19 and compare it with that of commonly used prognostic biomarkers. METHODS: Retrospective study of 128 COVID-19 in patients with no history of thyroid disease. Serum TSH, albumin, CRP, ferritin, and D-dimers were measured at admission. Outcomes were classified as "favorable" (discharge from hospital) and "adverse" (intubation or in-hospital death of any cause). The prognostic performance of TSH and other indices was assessed using binary logistic regression, machine learning classifiers, and ROC curve analysis. RESULTS: Patients with adverse outcomes had significantly lower TSH compared to those with favorable outcomes (0.61 versus 1.09 mIU/L, p < 0.001). Binary logistic regression with sex, age, TSH, albumin, CRP, ferritin, and D-dimers as covariates showed that only albumin (p < 0.001) and TSH (p = 0.006) were significantly predictive of the outcome. Serum TSH below the optimal cut-off value of 0.5 mIU/L was associated with an odds ratio of 4.13 (95% C.I.: 1.41-12.05) for adverse outcome. Artificial neural network analysis showed that the prognostic importance of TSH was second only to that of albumin. However, the prognostic accuracy of low TSH was limited, with an AUC of 69.5%, compared to albumin's 86.9%. A Naïve Bayes classifier based on the combination of serum albumin and TSH levels achieved high prognostic accuracy (AUC 99.2%). CONCLUSION: Low serum TSH is independently associated with adverse outcome in hospitalized Greek patients with COVID-19 but its prognostic utility is limited. The integration of serum TSH into machine learning classifiers in combination with other biomarkers enables outcome prediction with high accuracy.

Invasive methods for the diagnosis and management of intrathoracic extramedullary hematopoiesis: A literature review.

BACKGROUND: Extramedullary hematopoiesis is defined as hematopoiesis occurring outside of the bone marrow. It usually compensates insufficient bone marrow function or ineffective erythropoiesis and is observed mostly in hematological disorders. Most common locations of extramedullary hematopoiesis are the spleen, the liver and the lymph nodes. Intrathoracic extramedullary hematopoiesis is rare presenting as bilateral lobulated masses of lower paravertebral regions. This review summarizes the role of invasive techniques in the diagnosis and management of intrathoracic EMH and its complications. METHODS: An electronic search in PubMed and Google Scholar was conducted with the keywords "intrathoracic extramedullary hematopoiesis" AND "surgery" OR "video-assisted thoracic surgery (VATS)" OR "medical thoracoscopy" OR "biopsy" OR "thoracotomy" OR "image-guided biopsy" OR "median sternotomy", within 1970 to 2020 with the limitation of English language to include those articles reporting data on invasive techniques in intrathoracic extramedullary hematopoiesis. RESULTS: Overall, 93 articles were originally identified using our search criteria and from the reference list of the previously identified documents. Following elimination of duplicates, 29 were excluded after title, abstract or full text screening, since they did not report the use of invasive techniques in the diagnosis and management of intrathoracic extramedullary hematopoiesis. CONCLUSIONS: Although in some cases radiological features are typical for the diagnosis of intrathoracic extramedullary hematopoeisis, invasive methods such as bronchoscopy with transbronchial biopsy, ιmage-guided fine needle aspiration, endobronchial ultrasound-guided fine needle aspiration of the mass and mediastinoscopy, medical thoracoscopy, median sternotomy, video-assisted thoracoscopic surgery and thoracotomy, are essential for definite diagnosis and management.

A group of three fibroblast secreted polypeptides suppressed by cellular ageing and interferon-gamma.

The structural and quantitative characteristics of many fibroblast-secreted proteins are modified during the in vitro cellular ageing. Here we report that three polypeptides (80, 84 and 87 kDa) absent from late passage fibroblast cultures, are constitutively secreted from young (early passage) cultures of various fibroblast strains whereas they are suppressed by the action of interferon-gamma. The three polypeptides were isolated and monoclonal antibodies were produced against the 84 kDa polypeptide. Immunoprecipitation experiments showed that the three polypeptides share common epitopes. The 80 and 84 kDa polypeptides were studied further and proved to be glycosylated polypeptides exhibiting analogous CNBr digestion peptide maps and identity in their sequenced N-terminal segments.

Conjugation of acetylcholine receptor-protecting Fab fragments with polyethylene glycol results in a prolonged half-life in the circulation and reduced immunogenicity.

Antibodies to the acetylcholine receptor (AChR) cause AChR loss, resulting in the disease, myasthenia gravis (MG). The majority of the pathogenic antibodies seem to be directed against the main immunogenic region (MIR) of the AChR. In contrast to the intact antibodies, Fab fragments of anti-AChR antibodies are not themselves pathogenic and such fragments of anti-MIR monoclonal antibodies (mAbs) protect the AChR in vitro and in vivo against the pathogenic antibodies. However, Fab fragments have a very short in vivo half-life and are immunogenic, obstacles which must be overcome before their clinical use can be envisaged. We investigated the effect of conjugating Fab fragments to polyethylene glycol (PEG), a method known to increase the in vivo half-life and reduce the immunogenicity of proteins. When the Fab' fragments of two rat anti-MIR mAbs (nos. 35 and 195) were conjugated to methoxy-PEG-maleimide, the conjugates retained about 10% of their AChR binding activity and efficiently protected the AChR against the binding and modulating activity of myasthenic antibodies. Their in vivo half-life in rats was approximately 15 times longer than that of the unconjugated Fab' fragment and they were much less immunogenic in mice. This work represents an important step towards the clinical use of AChR-protective anti-MIR Fabs, but further improvements are needed before their clinical use is attempted.

Cellular ageing related proteins secreted by human fibroblasts.

Fibroblast secreted proteins participate in the formation of extracellular matrix. Extracellular matrix affects growth factor action, mediates cell adhesion and supports cell growth. Structural and quantitative characteristics of secreted proteins are modified in a similar manner, during both in vivo and in vitro cellular ageing. Such ageing related modifications may either be directly controlled by primary ageing causes, or evolve from a reformation of the extracellular matrix induced by a few ageing defects in key proteins such as fibronectin. They may result in the further inhibition of cell adhesion, cell stimulation by growth factors and, eventually, of cell proliferative ability.

Colonoscopy effects on serum prostate specific antigen levels.

INTRODUCTION: To evaluate the colonoscopy effects on serum levels of prostate specific antigen (PSA) and PSA ratio. SUBJECTS AND METHODS: Thirty men were studied (median age 68 years, range 32-89). All of them had their serum PSA (total and free) measured 24 hours prior to colonoscopy and also 24 hours, 7 and 30 days after procedure. RESULTS: 14 of 30 (47%) patients, had significantly (p = 0.045) increased Total PSA levels 24 hours after the procedure, 14 (47%) patients had insignificantly (p = 0.139) increased levels 7 days after, while 13 (43%) of them had insignificantly (p = 0.061) increased levels 30 days after colonoscopy. In 7 patients (23%), with total PSA levels in the "gray zone" (4-10 ng/ml) before colonoscopy, a near significant (p = 0.063) raise in PSA ratio was also observed 24 hours after. CONCLUSION: Flexible colonoscopy affect serum PSA (free and total) levels in certain patients. Special attention must be given to those patients with PSA levels in the "gray zone" before colonoscopy.

Influence of preoperative vesicle biopsy on the decision for radical prostatectomy.

PURPOSE: The presence of seminal vesicle invasion (SVI) by prostate cancer is difficult to detect clinically and is associated with poor prognosis. The aim of our study was to identify the efficacy of transrectal ultrasound-guided seminal vesicle biopsies in the detection of seminal vesicle invasion (SVI) in patients with prostate cancer. MATERIALS AND METHODS: One hundred transrectal ultrasound-guided seminal vesicle biopsies were performed in 50 patients with clinically localized prostate cancer. Every patient underwent two biopsies, one for each seminal vesicle. Radical retropubic prostatectomy was performed in all cases and the specimens with the attached seminal vesicles were examined for the presence of prostate cancer invasion. RESULTS: Of a total of 100 seminal vesical biopsies 87 were identified as seminal vesicle by characteristic epithelium. Cancer was found in 7 (8%) biopsies, confirmed in all cases by pathology in the surgical specimen. Eighty biopsies (40 patients) were normal. Pathological analysis of these 40 radical prostatectomy specimens revealed that 6 seminal vesicles (5 patients) were invaded by prostate cancer (6 false negative biopsies, 7.5%). Transrectal ultrasound images of 15 seminal vesicles were suspicious for invasion while 85 were normal. Of the 15 suspicious cases 11 were invaded by cancer (73.3%). Of the sonographically benign seminal vesicles 5 (5.88%) were invaded by cancer. Our data were analyzed by the ARCUS PRO-STAT statistical package. CONCLUSIONS: We suggest that transrectal ultrasound-guided seminal vesicle biopsy is useful and reliable for a more exact preoperative staging of prostate cancer, therefore helpful in correct decision making for radical prostatectomy.

DNA ploidy as a prognostic factor in muscle invasive transitional cell carcinoma of the bladder.

Radical cystectomy represents the treatment of choice for muscle-infiltrative bladder carcinoma; however, about 50% of patients relapse and die from the disease. In the present study, the prognostic significance of the DNA ploidy in transitional cell carcinoma of the urinary bladder (TCCB) is analyzed. The study was carried out on 66 patients with TCCB who underwent radical cystectomy. DNA ploidy was determined by flow cytometry (FCM) on paraffin-embedded specimens, and the results were analyzed and correlated with the tumor malignancy grade and stage and the clinical course. Forty of the 66 tumors studied (63%) were aneuploid. Aneuploid status was correlated with higher tumor T stage (P < 0.001) and grade (P < 0.001). Median follow up was 68 months (range: 12-105). Median survival was significantly longer in patients with diploid tumors (> 60 vs 45 months, P < 0.001). All patients with diploid tumors were alive and free of bladder cancer during follow-up, in contrast to only 30% of patients with aneuploid tumors. DNA ploidy was an independent prognostic factor, as shown by multivariate analysis (P = 0.006). All patients with pT > or = 3b and diploid tumors were alive at the time of analysis as opposed to none with aneuploid tumors. The results of this study suggest that DNA ploidy can provide prognostic information on patients with muscle invasive carcinoma of the bladder and might represent a means of selection for postoperative management.

Bacterial expression of a single-chain Fv fragment which efficiently protects the acetylcholine receptor against antigenic modulation caused by myasthenic antibodies.

Monoclonal antibodies (mAb) against the main immunogenic region (MIR) of the acetylcholine receptor (AChR) are very potent in inducing antigenic modulation of the AChR in animals and in muscle cell cultures. A recombinant antibody fragment of the rat anti-MIR mAb198 was cloned by polymerase chain reaction and expressed as soluble single-chain Fv fragment (scFv198) in E. coli and affinity purified. DNA sequencing was used to define the VH (IB) and VL (K2) chain gene usage. scFv198 was found immunologically and biologically active. Its binding affinity for the Torpedo AChR (KD = 2 +/- 0.6 nM) was very similar with that of the intact mAb198 (KD = 1.8 +/- 0.6 nM) while for the human AChR (KD = 80.7 +/- 16.6 nM) it was about four times lower than that of the intact mAb198 (KD = 21.6 +/- 6.6 nM). This fragment was capable of efficiently protecting the AChR in human cell cultures, against antigenic modulation caused by the intact mAb198 or by the antibodies from a myasthenic patient. The produced scFv198 fragment is, therefore, potentially useful in therapeutic applications for myasthenia gravis after appropriate genetic manipulations.

Expression of human-Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG).

The nicotinic AChR, a pentamer composed of alpha2betagamma(or epsilon)delta subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the alpha-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse-Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the alpha-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human alpha+ Torpedo betagammadelta AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the alpha-subunit. Interestingly, the anti-alpha-subunit antibodies predominated in low titre (0.6-7.4 nM) but not in high titre (10-386 nM) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-alpha-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the alpha-subunit.

The prognostic value of p53 and DNA ploidy following radical prostatectomy.

This study assesses the correlation of p53 immunoreactivity and DNA ploidy status with biochemical recurrence after radical prostatectomy. p53 protein expression and DNA ploidy were evaluated on 84 archival paraffin-embedded radical prostatectomy specimens. Patients were divided into two groups: those with low (38/84, 45%) and those with high (46/84, 55%) p53 immunoreactivity. The results were correlated with Gleason score, stage and serum PSA. Kaplan-Meier biochemical recurrence free survival and the Cox hazard-regression model were used for analysis. Multivariate analysis revealed p53, DNA ploidy, Gleason score, PSA and stage to be independent prognostic factors in this order. Kaplan-Meier analysis showed a significant difference in biochemical recurrence when p53 high expression and DNA aneuploidy were combined. The results of this study suggest that stratification for p53 expression and DNA ploidy status can provide additional prognostic information for patients with prostate carcinoma after radical prostatectomy.

Outcome after prenatal detection of a sporadic, unstable translocation t(5;21).

Amniotic fluid cultures from a 37 year old woman showed a sporadic 46,XX,t(5;21)(5qter----5p13 or p14::5pter----5p13 or p14::21p12----21qter) complement. In the majority of metaphases the 5p fragment was attached to the stalks of chromosome 21; however, in 9% of metaphases, the fragment was loosely attached by a 'thread' and in 6% it was completely detached. Silver staining and in situ hybridisation with a homologous ribosomal gene probe, which localises to stalk regions (nucleolar organisers, NOR) of human acrocentric chromosomes, failed to show a reciprocal exchange. Prognosis was uncertain because the possibility that the 5p fragment might have been lost in some cell lines could not be excluded. Nonetheless, the parents elected to continue the pregnancy. The translocation was confirmed in blood specimens obtained both at birth and at 1 year of age and showed similar instability. However, the proband shows no anomalies and is developing normally at 1 year.

Prognostic significance of CA 125, CD44, and epithelial membrane antigen in renal cell carcinoma.

OBJECTIVES: To study retrospectively CA 125, CD44, and epithelial membrane antigen (EMA) expression in renal cell carcinoma and their role as prognostic factors. CD44 is a cell adhesion molecule, and CA 125 and EMA are tumor-associated antigens used in the diagnosis and monitoring of the outcome and response to treatment of various human malignancies. Their expression and prognostic significance after resection of renal cell carcinoma have not been adequately studied. METHODS: The expression of CA 125, CD44, and EMA were studied immunohistochemically and correlated with the outcome of 92 patients who underwent nephrectomy for renal cell carcinoma. RESULTS: Positive staining was found for CA 125 in 28 patients (30.43%), CD44 in 48 patients (52.17%), and EMA in 74 patients (80.43%). CA 125 expression was increased in those with higher T stage (P <0.001) and histologic grade (P = 0.007). An inverse relationship was found between EMA expression and grade (P <0.001). The median follow-up was 41.5 months (range 30 to 65). The median survival for positive and negative patients was 34.6 versus 54.3 months for CA 125 (P = 0.0044), 48.3 versus 51.5 months for CD44 (P = 0.4677), and 53.2 versus 34 months for EMA (P = 0.0046). Multivariate analysis showed that CA 125 and EMA expression were independent prognostic factors (P = 0.021 and P = 0.018, respectively). Subgroup analysis showed that CA 125 expression predicted a significantly higher probability of death (28.6% versus 8%, P =0.0413) in patients with T1 or T2 tumors. CONCLUSIONS: CA 125 and EMA appear to be useful prognostic markers in renal cell carcinoma. Additional studies are needed to determine the value of these markers as a means of selection for postoperative management.