Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions.

To improve mesenchymal stem cell (MSC)-based therapy efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under microenvironmental stress conditions. We observed that human Wharton's jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-κß was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, formed under serum starvation stress. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. VTN is established as a key player which possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

Diagnostic Dilemma of Hemoglobinopathies Using High Performance Liquid Chromatography Alone: A Case Report from a Resource-Constrained Setting.

Hemoglobinopathies are quite common in India, and multiple awareness and screening initiatives exist for detection of thalassemia in the population. One of the most common and successfully used method for thalassemia screening is the high performance liquid chromatography (HPLC) test. However, in spite of its excellent usefulness as a screening tool, there are situations where HPLC alone may not be able to make an accurate diagnosis. Here we highlight a fairly common situation where HPLC alone failed to confirm the diagnosis. A detailed family and transfusion history along with clinical examination and investigations, such as a complete hemogram, HPLC, along with molecular studies would have aided in diagnosis. Another cause of concern raised by this case is that the most common mutation in our population, such as IVS-I-5 (G>C), HBB: c.92+5G>C, was not represented in the HPLC, and thus, was missed during the preconception screening process, leading to a chain of events.

Enzyme-free isolation of mesenchymal stem cells from decidua basalis of the human placenta.

Mesenchymal stem cells (MSCs), also referred to as "medicinal signaling cells," have gained prominence as candidates for cell-based therapy and in clinical trials owing to their regenerative and therapeutic properties. Here, we present a protocol for isolating MSCs from the decidua basalis layer of human placenta using an explant culture approach. We describe steps for collecting, disinfecting, and plating placental tissue. We then detail procedures for characterizing the isolated MSCs through flow cytometry and in vitro differentiation.

The Bethesda system for reporting thyroid fine needle aspirates: A study of 1763 patients; with histopathological correlations, in eastern India.

BACKGROUND: Fine-needle aspiration cytology (FNAC) is an important test for triaging patients with thyroid nodules. The 2007 National Cancer Institute Thyroid Fine-Needle Aspiration State-of-the-Science Conference helped instigate the recent publication of The Bethesda System for Reporting Thyroid Cytopathology (TBS). AIMS AND OBJECTIVES: Terminology and therefore the probability of malignancy vary between pathologists and institutions. The purpose of this study was to evaluate a single institution's experience to determine if sub-classification (neoplasm versus lesion) aids in identifying patients at higher risk for malignancy. Also, an effort in regional and worldwide variation of significance of adoption of Bethesda classification has been evaluated. MATERIALS AND METHODS: From 2018 to 2021, all patients with thyroid lesions' were evaluated for FNAC correlation with the surgical diagnosis. During this period, 1763 thyroid FNACs were reported. Histopathological examination (HPE) outcome data was available in 444 (25.2%) cases. RESULTS: The cytologic-histologic diagnostic discrepancy rate was 11.5%. The sensitivity and specificity of thyroid FNA for the diagnosis of malignancy were 71.4% and 49.5%, respectively. In our study, the maximum cases were benign (84.5%). The malignancy risk for the different categories in our study, as seen by follow-up HPE, has corroborated well with the Bethesda System. CONCLUSION: The current results indicated that FNAC provides an accurate diagnosis of thyroid malignancy. The classification is directly related to the risk of malignancy in each category and this helps in accurate clinical management of that category.

Pattern of autologous stem cell transplants at a tertiary care government hospital, with emphasis on transplant outcomes with pre-harvest CD34+ level.

Purpose: Autologous stem cell transplantation (ASCT) is an established therapy for many hematological diseases. This study assessed the pattern of ASCTs at a tertiary care center and associated factors, including pre-harvest CD34+ stem cell levels, leading to improved engraftment outcomes. Methodology: A retrospective study was conducted in India, between February 2009-August 2020. Patients who underwent ASCT for different hematological malignancies (n=65) were included, and the patients' age, sex, type and stage of disease, pre- and post-harvest CD34+ counts, and time to attain platelet/neutrophil engraftment or febrile neutropenia were analyzed. The post-harvest CD34+ dose was calculated. Pre-conditioning was performed using Granulocyte Colony Stimulating Factor (GCSF)±Plerixafor. Progression-free survival (PFS) was calculated using relapse/death as the endpoint. Results: The median age of the cohort (n=65) was 49 years, with a male preponderance. Multiple myeloma was the most common malignancy (70.8% [46/65]), requiring ASCT. The median time to ASCT was 13 months. All patients had received GCSF, while Plerixafor was used in 17 patients with a pre-harvest CD34+ count of <10 cells/µL. The median pre-harvest CD34+ concentration and post-harvest CD34+ cell dose was 27.54 cells/µL (n=26) and 5.23×106 cells/kg body weight (n=65), respectively. The median time to engraftment was 11 and 12 days, for neutrophils and platelets, respectively. One patient did not engraft and was excluded from the analysis. The time required to attain neutrophil engraftment was significantly lower (p=0.02) among freshly harvested stem cells (n=48) than that of cryopreserved products (n=17). Platelet engraftment associated with CD34+ pre- and post-harvest levels was not significant (p=0.06). The time to attain neutropenia and subsequent febrile neutropenia was significantly lower with an adequate post-harvest CD34+ dose (p=0.009). Febrile neutropenia was seen in 83.1% (54/65) patients. The median time for febrile neutropenia was 4 days post-ASCT. Pre- and post-harvest CD34+ concentrations were directly proportional to each other (p<0.001). The median PFS was 112 months (n=65). Survival was better in males (median PFS: 112 months) vs. females (median PFS: 59 months) (p=0.27). Eight patients relapsed, and eight patients had died. Conclusion: Although unrelated to age or sex, the post-harvest CD34+ dose was inversely related to febrile neutropenia. As pre- and post-harvest CD34+ levels were directly proportional, pre-harvest CD34+ concentrations may be reliably used to assess engraftment outcomes. Rapid neutrophil engraftment was noted in fresh stem cells with PFS of 112 months, and was better among males, the exact reason being unknown. Thus, a larger number of patients should be followed up to obtain an accurate picture.

Altered Adhesion and Migration of Human Mesenchymal Stromal Cells under Febrile Temperature Stress Involves NF-κß Pathway.

Mesenchymal stromal cells (MSCs) are clinically beneficial for regenerative treatment of chronic inflammation and autoimmune disorders. However, to attain maximum efficacy from the transplanted MSCs, evaluation of its interaction with the microenvironment, becomes critical. Fever being an important hallmark of inflammation, we investigated the effect of febrile temperature stress on adhesion and migration of umbilical cord-derived MSCs. 40 °C-exposure altered cellular morphology with significant cell flattening, delayed cell-matrix de-adhesion response and slower migration of MSCs, accompanied by suppressed directionality ratio and cell trajectory. Corresponding to the observed changes, mRNA expression of extracellular matrix genes like COLs and VTN were upregulated, while matrix metalloproteinase MMP-1, showed a significant downregulation. NF-κß pathway inhibition at 40 °C, led to reversal of gene expression pattern, cell spreading, de-adhesion dynamics and migration rate. Independent knockdown of p65 and p53 at 40 °C indicated inhibitory role of p65/p53/p21 axis in regulation of MMP-1 expression. P21 inhibits JNK activity, and JNK pathway inhibition at 40 °C resulted in further downregulation of MMP-1. Hence, our study provides the first evidence of cell migration getting adversely affected in MSCs under elevated temperature stress due to an inverse relationship between p65/p53/p21 and MMP1 with a possible involvement of the JNK pathway.

Isolation and Molecular Characterization of Progenitor Cells from Human Umbilical Cord.

Mesenchymal stem cells (MSCs) are multipotent precursor cells which have been isolated from different vascularized tissue sources. Due to their paracrine function of secreting trophic and immunomodulatory molecules, MSCs are successfully used in cell-based transplantations and provide an alternative medical paradigm for treating a variety of devastating disorders. Umbilical cord is a medical waste with a large, readily available donor pool. Since umbilical cord is a fetal tissue, MSCs derived from it are considered more primitive with proliferative and differentiation advantages over adult MSCs. We define here a simple, efficient, and reproducible protocol to isolate MSCs from WJ of human umbilical cord using a nonenzymatic procedure. Under the optimized culture conditions, the WJ-MSCs undergo robust proliferation, can be expanded up to 15-20 passages and express the characteristic MSC surface antigens. They can be differentiated into mesodermal lineages in vitro.

Spectrum of meningioma with special reference to prognostic utility of ER,PR and Ki67 expression.

BACKGROUND: Meningiomas are the most common primary central nervous system neoplasms originating from the arachnoid cap cells and constitute between 13% and 26% of all intracranial tumors. AIMS AND OBJECTIVES: The aim of the study was to analyze the age-, sex-, and site-wise distribution of different histological patterns of meningiomas seen in our center and to assess the status of estrogen receptor (ER), progesterone receptor (PR), and proliferation marker Ki-67 in various grades of meningioma. MATERIALS AND METHODS: A prospective study was done in 90 cases. Patients presented with symptoms of headache and seizure and underwent subsequent excision surgery at Neurosurgery Department were taken. We have studied histological typing and grading of the tumors, and immunohistochemical staining was done for ER, PR, and Ki-67. STATISTICAL ANALYSIS: Two-group comparison was done using Mann-Whitney U-test and Fisher's exact test. Comparison of Ki-67 expression between Grade 1 and Grade 2 meningiomas was determined using Mann-Whitney U-test. Comparison of ER and PR status between different histological grades was done by Fisher's exact test. Two-tailed P < 0.001 was considered statistically significant. RESULTS: According to histological type, meningothelial meningioma is most common (38.8%) followed by transitional (22.2%). PR positivity is seen in 96.34% of Grade 1 tumors, and all Grade 2 tumors were PR negative (Fisher's exact test P < 0.001). About 3.66% of Grade 1 and all Grade 2 tumors were positive for ER (Fisher's exact test two-tailed P < 0.001). Mean Ki-67 labeling index (LI) was 2.57 ± 1.674 among Grade I tumors, 7.11 ± 1.084 in Grade II meningiomas. CONCLUSIONS: Most of Grade 1 meningiomas show PRs positivity and lack of ERs positivity. Meningiomas with higher proliferation index and negative PR are very likely to be Grade II or Grade III. Evaluation of ER, PR status, and Ki-67 labeling index (LI) with histological evaluation helps us in providing information about the biologic behavior of meningiomas.

Diabetes insipidus as a presenting manifestation of Rathke's cleft cyst.

Rathke's cleft cysts (RCC) are cystic sellar and suprasellar lesions derived from remnants of Rathke's pouch, lined by cuboidal or columnar epithelium. RCC are usually asymptomatic but can present with headache, visual impairment, panhypopituitarism and hypothalamic dysfunction. Diabetes Insipidus as a presenting symptom of RCC is reported, but rare. We present a case of a 48-year-old male presenting with polyuria and on investigations found to have central diabetes insipidus due to a sellar RCC. Patient underwent transsphenoidal surgery with complete excision with resolution of his symptoms. His polyuria resolved post-surgery without vasopressin replacement, which has never been reported.