Antibody GD3G7 selected against embryonic glycosaminoglycans defines chondroitin sulfate-E domains highly up-regulated in ovarian cancer and involved in vascular endothelial growth factor binding

Chondroitin sulfate (CS) is abundantly present in the tumor stroma, and tumor-specific CS modifications might be potential targets to influence tumor development. We applied the phage display technology to select antibodies that identify these tumor-specific CS modifications. Antibody GD3G7 was selected against embryonic glycosaminoglycans, and it reacted strongly with CS-E (rich in GlcA-GalNAc4S6S units). In ovarian adenocarcinomas, strong expression of this CS-E epitope was found in the extracellular matrix, and occasionally on tumor cells. No expression was found in normal ovary and cystadenomas. Differential expression was found in ovarian carcinoma cell lines, which correlated with the gene expression of the GalNAc4S-6st enzyme, involved in biosynthesis of CS-E. Vascular endothelial growth factor (VEGF)-sensitive fenestrated (in normal tissues) and tumor blood vessels were both identified by antibody GD3G7, which might implicate a role for CS-E in VEGF biology. VEGF bound to CS-E and antibody GD3G7 could compete for binding of VEGF to CS-E. In conclusion, antibody GD3G7 identified rare CS-E-like structures that were strongly expressed in ovarian adenocarcinomas. This antibody might therefore be instrumental for identifying tumor-related CS alterations.     

Kinetics and Characterization of Non-enzymatic Fragmentation of Monoclonal Antibody Therapeutics

Purpose

To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same.

Methods

The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC).

Results

It was observed that 57% of the mAb product fragmented over 4 days into two fragment species – Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively.

Conclusion

High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.

     

Primary vs delayed primary closure in patients undergoing lower limb amputation following trauma: A randomised control study

Lower limb crush injury is a major source of mortality and morbidity in trauma patients. Complications, especially surgical site infections (SSIs) are a major source of financial burden to the institute and to the patient as it delays rehabilitation. As such, every possible attempt should be made to reduce any complications. We, thus, aimed to compare the outcomes in early vs delayed closure of lower extremity stumps in cases of lower limb crush injury requiring amputation, so as to achieve best possible outcome. A randomised controlled study was conducted in the Division of Trauma Surgery & Critical Care at Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi from 1 September 2018 to 30 June 2019 and included patients undergoing lower limb amputation below hip joint. Patients were randomised in two groups, in one group amputation stump was closed primarily, while in the second group delayed primary closure of stump was performed. We compared rate of SSI, length of hospital stay, and number of surgeries in both the groups. Fifty‐six patients with 63 amputation stumps were recruited in the study. Mean age of patients in the study was 34 years, of which about 95% patients were males. The most common mechanism of injury was road traffic injury in 66% of patients. Mean injury severity score was 12.28 and four patients had diabetes preoperatively. Total 63 extremities were randomised with 30 cases in group I and 33 cases in group II as per computer‐generated random number. Above knee amputations was commonest (57.14%) followed by below knee amputations (33.3%). Two patients died in the current study. In group I, In‐hospital infection was detected in 7 cases (23.3%) and in group II 9 cases (27.3%) had SSI during hospital admission (P > .05). Mean hospital stay in group I was 10.32 ± 7.68 days and in group II was 11 ± 8.17 days (P > .05). Road traffic injuries and train‐associated injuries are a major cause of lower limb crush injuries, leading to limb loss. Delayed primary closure of such wounds requires extra number of surgical interventions than primary closure. There is no difference in extra number of surgical interventions required in both the groups. Thus, primary closure can be safely performed in patients undergoing lower limb amputations following trauma, provided that a good lavage and wound debridement is performed.     

Hepatitis C Virus Epitope Immunodominance and B Cell Repertoire Diversity

Despite the advent of effective, curative treatments for hepatitis C virus (HCV), a preventative vaccine remains essential for the global elimination of HCV. It is now clear that the induction of broadly neutralising antibodies (bNAbs) is essential for the rational design of such a vaccine. This review details the current understanding of epitopes on the HCV envelope, characterising the potency, breadth and immunodominance of antibodies induced against these epitopes, as well as describing the interactions between B-cell receptors and HCV infection, with a particular focus on bNAb heavy and light chain variable gene usage. Additionally, we consider the importance of a public repertoire for antibodies against HCV, compiling current knowledge and suggesting that further research in this area may be critical to the rational design of an effective HCV vaccine.     

Left atrial abnormality by electrocardiogram predicts left ventricular hypertrophy by echocardiography in the presence of right bundle-branch block

Background: Left ventricular hypertrophy (LVH) on the electrocardiogram (ECG) may be masked in the presence of complete right bundle-branch block (RBBB). Left bundle-branch block on the ECG is associated with LVH at autopsy in 93% of hearts studied. However, RBBB does not predict LVH and the usual ECG criteria applied for LVH may not be reliable in the presence of RBBB. Hypothesis: The study was undertaken to evaluate left atrial (LA) abnormality as a criterion for the diagnosis of LVH in the presence of RBBB. Methods: Left atrial abnormality in the ECG was assessed by two independent observers as a criterion of LVH in the presence of RBBB in 100 patients, and data were compared with those of 50 patients without LA abnormality. Results: Left ventricular hypertrophy was confirmed by echocardiographic determination of left ventricular (LV) mass in both groups. Observers reliably differentiated between hy-pertrophied and normal-sized LV in the presence of RBBB by using LA abnormality as an ECG criterion when correlated with LV mass determined by echocardiography. Observer 1 correctly detected LVH in 88% and Observer 2 in 82% of patients. False positive diagnosis was made in 12 and 18% of patients by Observers 1 and 2, respectively. Observers' performance of recognition of LA abnormality in the present study was 94%. Results showed sensitivity of 76 and 70% and specificity of 84 and 92% for Observers 1 and 2, respectively. Left ventricular mass increased significantly and was diagnostic of LVH in 92% of patients with LA abnormality. Left ventricular mass was high in 84% of patients when corrected by body surface area. LVH in the presence of RBBB by the ECG was found in only seven patients (5%) when six commonly used conventional criteria of diagnosis of LVH by ECG were employed. Regression analysis found LA abnormality to be a strong independent predictor of increased LV mass. Multiple regression analysis revealed that age, body mass index, body surface area, and frontal axis are also significant predictors of LV mass. Conclusion: The results obtained by the correlation of LA abnormality by ECG and LVH by echocardiography conclude that LA abnormality by ECG was significantly diagnostic of LV hypertrophy in the presence of RBBB.     

Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes

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Treatment of non-union of humerus diaphyseal fractures: a prospective study comparing interlocking nail and locking compression plate

Background

The aim of this prospective comparative study was to compare outcomes and complications of humeral diaphyseal fracture non-unions managed with humerus interlocking nail (HIL) and locking compression plate (LCP).

Materials and methods

40 patients with non-union of humeral diaphyseal fractures were included in this study and were randomly allocated in two groups; group A had 20 cases treated with HIL and group B had 20 cases treated with LCP. Clinico-radiological assessments were done for each case up to 2-year follow-up period. Primary outcome measures (time to fracture union, union rate) and secondary outcome measures (functional outcome and complication such as infection, malunion, delayed union, implant failure, joint stiffness and iatrogenic radial nerve palsy) were compared between both the groups. Disabilities of the arm, shoulder and hand (DASH) scoring and Steward and Hundley’s scoring system were used to assess functional outcome of the fracture fixation.

Results

There was no significant difference (p = 0.12) in terms of mean fracture union time between group A (15.8 ± 4.2 weeks) and group B (17.2 ± 3.8 weeks). Group A had 95 % union rate and group B had 100 % union rate (p = 0.14). At the 2-year follow-up visit, there was no significant difference found between both the groups regarding range of motion of shoulder and elbow joint. There was no significant difference found in final functional outcomes between both the groups on comparing DASH score (p = 0.14) and Steward and Hundley’s score (p = 0.08). In terms of complications, there was insignificant difference found between both the groups.

Conclusions

This study concludes that both the implants can be used in non-union of humeral shaft fractures with good functional outcomes and acceptable rate of complications.     

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

Primary pancreatic lymphoma-diagnosed on computed tomography: A rare case report.

Primary pancreatic lymphoma is a rare disease. It comprises less than 0.5 % of pancreatic neoplasm and 0.1% of malignant lymphoma. It should be differentiated from pancreatic adenocarcinoma because management differs. At computed tomography, 2 types of morphology of primary pancreatic lymphoma have been described- a localized well-circumscribed tumoral form and another diffuse enlargement infiltrating or replacing most of the pancreatic gland. Here, we are presenting computed tomography (CT) imaging features of a case of primary pancreatic lymphoma in a 27 year old female who presented with a complaint of abdominal pain radiating to the back for 3 months and yellowish discoloration of sclera and skin for the last 15 days. In contrast-enhanced computed tomography an exophytic homogenously hypoenhancing mass arising from head and neck region of pancreas was seen. Involvement of common bile duct (CBD) and duodenum was there. The main pancreatic duct was not dilated. Common hepatic artery was encased by mass without arterial luminal narrowing or distortion. Diagnosis of primary pancreatic lymphoma was suggested on basis of imaging findings and further confirmed with fine-needle aspiration cytology.