Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzmes A and B in the cytotoxic response in vivo

Evaluation of semiautomated internal carotid artery stenosis quantification from 3-dimensional contrast-enhanced magnetic resonance angiograms

Rationale and Objectives: The performance of a semiautomatic technique for internal carotid artery (ICA) stenosis quantification of the internal carotid artery in contrast-enhanced magnetic resonance angiography was evaluated. Materials and Methods: The degree of stenosis of 52 ICAs was quantified by measuring the cross-sectional area along the center lumen line. This was performed both by 3 independent observers and the semiautomated method. The degree of stenosis was defined as the amount of cross-sectional lumen reduction. Results: Agreement between the method and observers was good (weighted-kappa, kappa(w) = 0.89). Reproducibility of measurements of the semiautomated technique was better (kappa(w) = 0.97) than that of the observers (kappa(w) = 0.76), and the evaluated technique was considerably less time-consuming. Conclusions: Because the user interaction is limited, this technique can be used to replace an expert observer in 3-dimensional stenosis quantification of the ICA at CE-MRA in clinical practice

Limitations of Dutch Growth Research Foundation Commercial Software Weight Velocity for Age Standard Deviation Score

BACKGROUND The commercial software for hospitals, Weight Velocity for Age Standard Deviation Score (SDSWVA), claims to document the growth and development of children, although published details are unavailable. The statistics-derived parameter SDSWVA includes the weight velocity at age t, WV(t) (weight gained between t and (t-1.23) years, divided by 1.23), and 3 standard weight velocity curves at average age AA, defined as AA=t-1.23/2 years. SDSWVA denotes the number of standard deviations that WV(t) deviates from the 0 SD weight velocity at AA. WV(t) yielded erroneous outcomes when applied to weights of a seriously underweight boy with an allergy to cows' milk who showed strong weight growth after being fed on food free of cows' milk. The SDSWVA software tacitly suggests that it is more accurate than WV(t). CASE REPORT The case of this boy was previously described in this Journal. Using SDSWVA(t,AA) software, his weight growth was analyzed by his third pediatrician, beginning at age 1.5 years. The diagnosis of the mother with Pediatric Condition Falsification was confirmed, adding 6 months to foster care, which totalled 8.5 months. Testing of the SDSWVA software on the boy's weight curve yielded results that were complex, nontransparent, and as erroneous as WV(t), explaining the misdiagnosis by the third pediatrician. CONCLUSIONS SDSWVA software should not be used for children under 3 years and during variable weight behavior. Erroneous performance, unpublished details, and an error identified in their new but untested software make the Dutch Growth Research Foundation unlikely to meet the 2020 European Union regulations for in vitro medical devices

Detailed examination of lymph nodes improves prognostication in colorectal cancer

Up to 30% of stage II patients with curatively resected colorectal cancer (CRC) will develop disease recurrence. We evaluated whether examination of lymph nodes by multilevel sectioning and immunohistochemical staining can improve prognostication. Lymph nodes (n = 780) from 36 CRC patients who had developed disease recurrence (cases) and 72 patients who showed no recurrence of disease for at least 5 years (controls) were analyzed. Sections of 4 levels at 200-mu m interval were immunohistochemically stained for cytokeratin expression. The first level was analyzed by conventional and automated microscopy, and the 3 following levels were analyzed by automated microscopy for the presence of tumor cells. Overall, cases showed more micrometastases (3 patients) than controls (1 patient). Analysis of a second level led to the additional detection of 1 patient with micrometastases (case) and 1 patient with macrometastasis (case). Examining more levels only led to additional isolated tumor cells, which were equally divided between cases and controls. Likewise, automated microscopy resulted only in detection of additional isolated tumor cells when compared with conventional microscopy. In multivariate analysis, micrometastases [odds ratio (OR) 26.3, 95% confidence interval (CI) 1.9-364.8, p = 0.015], T4 stage (OR 4.8, 95% CI 1.4-16.7, p = 0.013) and number of lymph nodes (OR 0.9, 95% CI 0.8-1.0, p = 0.028) were independent predictors for disease recurrence. Lymph node analysis of 2 levels and immunohistochemical staining add to the detection of macrometastases and micrometastases in CRC. Micrometastases were found to be an independent predictor of disease recurrence. Isolated tumor cells were of no prognostic significance.</p

Limitations of Weight Velocity Analysis by Commercial Computer Program Growth Analyser Viewer Edition

Commercial software package “Growth Analyser Viewer Edition” (“GAVE”) aims to document, monitor and analyze growth and development in children and adolescents. Although its clinical and scientific use is widespread, there are no published studies that describe the method and its validation. We were informed that GAVE calculates the weight velocity (kg/year) at age t from the weight difference between t and 448 days earlier or at birth, divided by the time difference. We recently discussed a case of false child abuse diagnosis (Pediatric Condition Falsification), resulting in the separation of the child from its parents, in which GAVE played a negative contributing role. To prevent such inappropriate diagnoses, we analyzed GAVE from a schematic representation of the measured clinical weight curve, with precisely defined weight velocities. In conclusion, the 448 days included for weight velocity predictions by GAVE caused the erroneous outcomes. Until the necessary changes to the software are implemented and validated, we advise against the use of GAVE in infants younger than 1.5 years, if multiple weight changes occur within 448 days, and following a long-lasting weight velocity change. Our analysis suggests to discard all medical software packages that lack public description and proof of validation

Cancer risk in children, adolescents, and young adults conceived by ART in 1983-2011

STUDY QUESTION: Do children, adolescents, and young adults born after ART, including IVF, ICSI and frozen-thawed embryo transfer (FET), have an increased risk of cancer compared with children born to subfertile couples not conceived by ART and children from the general population? SUMMARY ANSWER: After a median follow-up of 18 years, the overall cancer risk was not increased in children conceived by ART, but a slight risk increase was observed in children conceived after ICSI. WHAT IS KNOWN ALREADY: There is growing evidence that ART procedures could perturb epigenetic processes during the pre-implantation period and influence long-term health. Recent studies showed (non-)significantly increased cancer risks after ICSI and FET, but not after IVF. STUDY DESIGN, SIZE, DURATION: A nationwide historical cohort study with prospective follow-up was carried out, including all live-born offspring from women treated with ART between 1983 and 2011 and subfertile women not treated with ART in one of the 13 Dutch IVF clinics and two fertility centers. PARTICIPANTS/MATERIALS, SETTING, METHODS: Children were identified through the mothers' records in the Personal Records Database. Information on the conception method of each child was collected through the mother's medical record. In total, the cohort comprises 89 249 live-born children of subfertile couples, of whom 51 417 were conceived using ART and 37 832 were not (i.e. conceived naturally, through ovulation induction, or after IUI). Cancer incidence was ascertained through linkage with the Netherlands Cancer Registry for the period 1989-2019. Cancer risk in children conceived using ART was compared with risk in children born to subfertile couples but not conceived by ART (hazard ratio (HR)) and children from the general population (standardized incidence ratios (SIRs)). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 358 cancers were observed after a median follow-up of 18 years. Overall cancer risk was not increased in children conceived using ART, when compared with the general population (SIR = 0.96, 95% CI = 0.81-1.12) or with children from subfertile couples not conceived by ART (HR = 1.06, 95% CI = 0.84-1.33). Compared with children from subfertile couples not conceived by ART, the use of IVF or FET was not associated with increased cancer risk, but ICSI was associated with a slight risk increase (HR = 1.58, 95% CI = 1.08-2.31). Risk of cancer after ART did not increase at older ages (≥18 years, HR = 1.26, 95% CI = 0.88-1.81) compared to cancer risk in children not conceived by ART. LIMITATIONS, REASONS FOR CAUTION: The observed increased risk among children conceived using ICSI must be interpreted with caution owing to the small number of cases. WIDER IMPLICATIONS OF THE FINDINGS: After a median follow-up of 18 years, children conceived using ART do not have an increased overall cancer risk. Many large studies with prolonged follow-up are needed to investigate cancer risk in (young) adults conceived by different types of ART. In addition, international pooling of studies is recommended to provide sufficient power to study risk of specific cancer sites after ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by The Dutch Cancer Society (NKI 2006-3631) that funded the OMEGA-women's cohort, Children Cancer Free (KIKA; 147) that funded the OMEGA-I-II offspring cohort. The OMEGA-III offspring cohort was supported by a Postdoc Stipend of Amsterdam Reproduction &amp; Development, and the Eunice Kennedy Shriver National Institute of Child Health &amp; Human Development of the National Institutes of Health under Award Number R01HD088393. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare no competing interests. TRIAL REGISTRATION NUMBER:N/A.</p

Effect of long-term voluntary exercise wheel running on susceptibility to bacterial pulmonary infections in a mouse model

Regular moderate exercise has been suggested to exert anti-inflammatory effects and improve immune effector functions, resulting in reduced disease incidence and viral infection susceptibility. Whether regular exercise also affects bacterial infection susceptibility is unknown. The aim of this study was to investigate whether regular voluntary exercise wheel running prior to a pulmonary infection with bacteria (P. aeruginosa) affects lung bacteriology, sickness severity and phagocyte immune function in mice. Balb/c mice were randomly placed in a cage with or without a running wheel. After 28 days, mice were intranasally infected with P. aeruginosa. Our study showed that regular exercise resulted in a higher sickness severity score and bacterial (P. aeruginosa) loads in the lungs. The phagocytic capacity of monocytes and neutrophils from spleen and lungs was not affected. Although regular moderate exercise has many health benefits, healthy mice showed increased bacterial (P. aeruginosa) load and symptoms, after regular voluntary exercise, with perseverance of the phagocytic capacity of monocytes and neutrophils. Whether patients, suffering from bacterial infectious diseases, should be encouraged to engage in exercise and physical activities with caution requires further research

The EORTC-DeCOG nomogram adequately predicts outcomes of patients with sentinel node-positive melanoma without the need for completion lymph node dissection

Purpose: Based on recent advances in the management of patients with sentinel node (SN)–positive melanoma, we aimed to develop prediction models for recurrence, distant metastasis (DM) and overall mortality (OM). Methods: The derivation cohort consisted of 1080 patients with SN-positive melanoma from nine European Organization for Research and Treatment of Cancer (EORTC) centres. Prognostic factors for recurrence, DM and OM were studied with Cox regression analysis. Significant factors were incorporated in the models. Performance was assessed by discrimination (c-index) and calibration in cross-validation across centres. The models were externally validated using a prospective cohort consisting of 705 German patients with SN-positive: 473 trial participants of the German Dermatologic Cooperative Oncology Group study (DeCOG-SLT) and 232 screened patients. A nomogram was developed for graphical presentation. Results: The final model for recurrence and the calibrated models for DM and OM included ulceration, age, SN tumour burden and Breslow thickness. The models showed reasonable calibration. The c-index for the recurrence, DM and OM model was 0.68, 0.70 and 0.70, respectively, and 0.70, 0.72 and 0.74, respectively, in external validation. The EORTC-DeCOG model identified a robust low-risk group, with all identified low-risk patients (approximately 4% of the entire population) having a 5-year recurrence probability of <25% and an overall 5-year recurrence rate of 13%. A model including information on completion lymph node dissection (CLND) showed only marginal improvement in model performance. Conclusions: The EORTC-DeCOG nomogram provides an adequate prognostic tool for patients with SN-positive melanoma, without the need for CLND. It showed consistent results across validation. The nomogram could be used for patient counselling and might aid in adjuvant therapy decision-making