FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

INTRODUCTION: Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. METHODS: EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. RESULTS: A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). CONCLUSION: Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Preclinical development of 1B7/CD3, a novel anti-TSLPR bispecific antibody that targets CRLF2-rearranged Ph-like B-ALL.

Patients harboring CRLF2-rearranged B-lineage acute lymphocytic leukemia (B-ALL) face a 5-year survival rate as low as 20%. While significant gains have been made to position targeted therapies for B-ALL treatment, continued efforts are needed to develop therapeutic options with improved duration of response. Here, first we have demonstrated that patients with CRLF2-rearranged Ph-like ALL harbor elevated thymic stromal lymphopoietin receptor (TSLPR) expression, which is comparable with CD19. Then we present and evaluate the anti-tumor characteristics of 1B7/CD3, a novel CD3-redirecting bispecific antibody (BsAb) that co-targets TSLPR. In vitro, 1B7/CD3 exhibits optimal binding to both human and cynomolgus CD3 and TSLPR. Further, 1B7/CD3 was shown to induce potent T cell activation and tumor lytic activity in both cell lines and primary B-ALL patient samples. Using humanized cell- or patient-derived xenograft models, 1B7/CD3 treatment was shown to trigger dose-dependent tumor remission or growth inhibition across donors as well as induce T cell activation and expansion. Pharmacokinetic studies in murine models revealed 1B7/CD3 to exhibit a prolonged half-life. Finally, toxicology studies using cynomolgus monkeys found that the maximum tolerated dose of 1B7/CD3 was ≤1 mg/kg. Overall, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 in patients with CRLF2-rearranged B-ALL.

Prognostic significance of matrix metalloproteinase 9 in COMET operated chronic ocular Stevens-Johnson syndrome.

PURPOSE: Molecular pathogenesis underlying persistent ocular surface inflammation in chronic Stevens-Johnson syndrome (SJS) still remains largely unexplored. The present study investigates the expression of matrix metalloproteinase 2 (MMP2), MMP3, MMP9, MMP11 and TIMP1 (tissue inhibittor of matrix metalloproteinase 1) in pannus tissues of chronic ocular SJS undergoing cultivated oral mucosal epithelial transplantation (COMET) and their prognostic relevance. METHODS: In this prospective study, 45 eyes with chronic SJS underwent COMET for visual and anatomical rehabilitation. Preoperative and postoperative clinical parameters were documented. MMP2, MMP3, MMP9, MMP11 and TIMP1 expression were assessed using immunohistochemistry and quantitative real time PCR. Inflammadry MMP9 assay was performed at 1-year follow-up. Kaplan-Meier curves and Cox proportional hazard models were used to correlate protein expression with clinicopathological parameters and COMET graft survival outcomes. RESULTS: MMP9 and MMP11 positivity was seen in both pannus epithelia (48% and 55%, respectively) and in stromal layer (57% and 33%, respectively) while MMP2 and MMP3 showed only pannus epithelial positivity in 35% and 51% cases, respectively. High MMP9 stromal expression was significantly associated with preoperative corneal keratinisation (p=0.011), conjunctival hyperaemia (p=0.014), symblepharon (p=0.028). High MMP9 and MMP3 epithelial expression were found to be independent risk factors for poor best-corrected visual acuity (BCVA) outcomes post-COMET (p=0.022 and p=0.048). Multivariate analysis revealed MMP9 to be the best prognostic marker (p=0.050). CONCLUSION: Our findings suggest that differential expression of MMPs and TIMP1 is seen in SJS in chronic stage. Emergence of MMP9 as a poor prognostic predictor of BCVA post COMET and postoperative MMP9 immunoassay positivity could be a useful tool in further studies to understand the unresolved ocular surface inflammation seen in SJS.

Prenatal diagnosis for hemophilia A (intron 22 inversion) reveals a rare association with Klinefelter syndrome with diagnostic difficulties in molecular interpretation.

Hemophilia A is an X-linked recessive disorder caused by genetic abnormalities in the F8 . Klinefelter syndrome is sex chromosome aneuploidy caused by nondisjunction during meiosis in the germ cells or mitotic cell divisions in the early embryonic cells. We here report an intriguing case of a prenatal diagnosis where a rare association of hemophilia A and Klinefelter syndrome was found in a fetus. This case highlights the diagnostic difficulty where the inverse-PCR for intron 22 inversion defect leading to hemophilia A did not amplify. Indirect molecular testing was done using multiallelic extragenic variable number tandem repeat (VNTR) DXS52 (St14) and polymorphic markers. The interpretation was further complicated by the presence of Klinefelter syndrome. This case highlights the challenges faced when such rare combinations are found during prenatal diagnosis.

Indian traditional medicinal plants in ophthalmic diseases.

Objective: Traditional herbal plants have been in use since ancient times to treat ophthalmic conditions; so, the aim of this study is to evaluate some potent Indian traditional medicinal plants used in ophthalmic diseases in order to summarize their potential effect in ophthalmology along with their mechanism of action. Materials and Methods: Databases PubMed, Google Scholar, and Embase were extensively explored. Additionally, relevant textbooks and literatures were consulted to summarize most of the considerable scientific literature for the review. Search term included ophthalmology, glaucoma, cataract, trachoma, conjunctivitis, traditional medicines, Unani drugs, and ayurvedic drugs were used. Around 80 review articles were consulted from the year 1982 to 2021. Results: The traditional medicinal plants are easily available, cost-effective and have no associated side effects in comparison to current conventional treatments. Moreover, these drugs in oppose to modern medicine, have an inherent potential to accelerate the body's own immunity to fight against any infection. A large volume of scientific studies has reported the beneficial effects of traditional drugs in ophthalmology. Conclusion: This review, therefore, describes the potential benefits and uses of some traditional medicinal plants used in ophthalmic diseases.

The protective role of HSP27 in ocular diseases.

Heat shock proteins (HSPs) are stress-induced proteins that are important constituents of the cell's defense system. The activity of HSPs enhances when the cell undergoes undesirable environmental conditions like stress. The protective roles of HSPs are due to their molecular chaperone and anti-apoptotic functions. HSPs have a central role in the eye, and their malfunction has been associated with the manifestation of ocular diseases. Heat shock protein 27 (HSP27, HSPB1) is present in various ocular tissues, and it has been found to protect the eye from disease states such as retinoblastoma, uveal melanoma, glaucoma, and cataract. But some recent studies have shown the destructive role of HSP27 on retinal ganglionic cells. Thus, this article summarizes the role of heat shock protein 27 in eye and ocular diseases and will focus on the expression, regulation, and function of HSP27 in ocular complications.

Achondroplasia-First Report from India of a Rare FGFR3 Gene Variant.

The clinical manifestations of FGFR3 sequence variations can vary from mild unnoticed short stature to neonatal lethal dwarfism and can be causative of phenotypes including achondroplasia, hypochondroplasia, and thanatophoric dysplasia. Clinical data describe an 11 month old girl with restricted growth and preserved intellect. She had rhizomelic short stature with peculiar facies but no Acanthosis nigricans. In view of the absence of the hotspot mutation c.1138 G>A/G>C (p.Gly380Arg), complete gene sequencing was done that revealed a rare sequence variation, NM_000142.4:c.1043C>G (p.Ser348Cys) in FGFR3. This sequence variation has not been reported from India so far. This report emphasizes the benefit of sequencing the whole gene in individuals who are negative for hotspot mutation of achondroplasia with strong clinical suspicion.

The mouse double minute 2 309T>G polymorphism and retinoblastoma risk: A meta-analysis.

PURPOSE: Mouse double minute 2 (MDM2) homolog is a protein that in humans is encoded by the MDM2 gene. It is expressed in retinoblastoma (Rb) cells and acts as a key negative regulator of the p53 tumor suppressor gene. Several studies have investigated the association of Rb with MDM2 309T>G polymorphism, but the results were conflicting. To derive a more precise estimation of the association, we performed a meta-analysis of the relationship between MDM2 309T>G polymorphism with Rb in all published studies. METHODS: Published literature from PubMed and other databases were retrieved. All the reported studies evaluating the association between MDM2 309T>G polymorphism and Rb risk were included. The pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using the fixed-effect model. A total of four case-control studies, including 520 cases and 745 controls were included. RESULTS: This meta-analysis found that MDM2 309T>G polymorphism was significantly associated with Rb risk in the dominant model, TG+GG versus TT (OR = 1.43, 95% CI = 1.11-1.84, P = 0.006). CONCLUSION: The present meta-analysis suggested that MDM2 309T>G polymorphism has a significant association with increased Rb risk.

Comparative Evaluation of Pregabalin and Clonidine as Preemptive Analgesics for the Attenuation of Postoperative Pain Following Thoracolumbar Spine Surgery.

STUDY DESIGN: Prospective, randomized, double blind, placebo-controlled study. PURPOSE: To compare clonidine and pregabalin with placebo for the attenuation of postoperative pain after thoracolumbar spinal surgery and instrumentation. OVERVIEW OF LITERATURE: Spine surgery is associated with moderate to severe postoperative pain that needs to be controlled to improve patient's outcome. Alpha 2 agonists (e.g., clonidine) and gabapentenoids (e.g., pregabalin) are successfully used as part of a multimodal analgesic regimen. METHODS: Total 75 patients were enrolled and randomly allocated into three groups. Group P received pregabalin (150 mg), group C received clonidine (150 mcg), and group N received placebo 90 minutes preoperatively. A standard anesthesia protocol comprising fentanyl, thiopentone, vecuronium, nitrous oxide, and oxygen in isoflurane was used for all patients. Postoperative recovery profile, pain, time for first analgesic, 24-hour analgesic requirement, sedation, and hemodynamic parameters were noted. RESULTS: Recovery profile was similar in all three groups; however, the patients in group P and C were more sedated (p<0.05). Group N patients had a higher Visual Analog Scale (VAS) score (p<0.05) and the time for first analgesic was also lower (p=0.02). Postoperative (24-hour) analgesic requirement was maximum in group N, followed by that in group C and group P. The VAS score was highest in the control group; however, after 12 hours, it was similar in all groups. CONCLUSIONS: Postoperative pain and analgesic requirement is significantly attenuated by preoperative administration of a single dose of clonidine (150 mcg) or pregabalin (150 mg); pregabalin was more effective. Thus, their use offers a reasonable strategy for pain management in patients undergoing spine surgery.

Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome.

Inflammation in uveal melanoma (UM) is linked to a bad prognosis. It is rare type of cancer, of which the metastases are usually fatal within a year. Infiltration with an inflammatory infiltrate increases with disease progression but does not seem to inhibit metastasis. The Canonical NFκB (C-NFκB) pathway is known to play a crucial role in tumor inflammation. We therefore, studied the expression of canonical NFκB proteins and their prognostic relevance in UM. Our study evaluated the expression of C-NFκB proteins (p65, p50, and c-Rel) by using immunohistochemistry on sections from 75 formalin-fixed UM. Activation of the NFκB subunit was determined on fresh tumor specimens by measuring the DNA-binding activity in nuclei using an NFκB ELISA assay. Real-time PCR was performed on frozen material on 58 tumors. The presence of native C-NFκB heterodimers (p65/p50 and c-Rel/p50) was confirmed by co-immunoprecipitation followed by Western blotting. We observed a high nuclear immunoreactivity of p65, p50, and c-Rel proteins in 54, 60 and 41% UM cases, respectively. Expression of C-NFκB proteins significantly correlated with parameters which are related to the inflammatory environment of UM. Nuclear immunoreactivity of p65 and p50 was associated with lower patient survival (p = 0.041; p = 0.048) while c-Rel was not. Our finding reveals that C-NFκB proteins expressed are more often in UM with inflammation than those without inflammation. Activation of the canonical NFκB pathway is more frequent in high risk UM patients. These observations might help to understand the behaviour of high risk tumors, with upregulation of C-NFκB proteins contributing to tumor aggressiveness.

Possible role of differentially expressing novel protein markers (ligatin and fibulin-7) in human aqueous humor and trabecular meshwork tissue in glaucoma progression.

The pathological mechanism underlying glaucoma has always been a complex aspect of this permanently blinding disease but proteomic studies have been helpful in elucidating it to a great extent in several studies. This study was designed to evaluate the expression and to get an idea about the function of two novel markers (ligatin and fibulin-7) identified in human aqueous humor (hAH) in relation to glaucomatous progression. A significant increase in the protein content of glaucomatous hAH compared to that of non-glaucomatous controls (NG-Ctrls) was observed. Ligatin, fibulin-7, and its proteolysis were revealed in hAH of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG) and NG-Ctrls. Quantification confirmed no significant difference in expression of ligatin, whereas fibulin-7 was significantly (P < 0.05) low in hAH of PACG in comparison to NG-Ctrls and POAG. Importantly the immunohistochemical assay for both indicated their possible involvement in the maintenance of the appropriate structure of TM in vivo. Since oxidative stress is a major contributor to glaucomatous pathogenesis, in vitro analysis of nuclear and cytoplasmic fractions indicated intracellular changes in localization and expression of ligatin upon oxidative insult of human trabecular meshwork (TM) cells. While no such changes were found for fibulin-7 expression. This was also corroborated with the immunocytochemical assay. Though a study with a small sample size, this is the first report which confirms the presence of ligatin and fibulin-7 in hAH, quantified their differential expression, and indicated the possibility of their involvement in the maintenance of the TM structure.

Comparative evaluation of the aqueous humor proteome of primary angle closure and primary open angle glaucomas and age-related cataract eyes.

PURPOSE: To analyze and compare the total proteome of aqueous humor (AH) from patients having primary angle closure glaucoma (PACG), primary open angle glaucoma (POAG) and age-related cataract. MATERIALS AND METHODOLOGY: Aqueous humor was collected from age-matched PACG, POAG and cataract patients who underwent surgery, and it was immediately stored at - 80 °C until analysis. From each sample, 25 µg of total protein was subjected to trypsin digestion and subsequently LC-MS/MS analysis was performed for the deep proteome analysis. The data acquired after the LC-MS/MS analysis were analyzed using Proteome Discoverer 1.4. The identified peptide matches were validated using percolator, at less than 1% false discovery rates. RESULTS: A total of 625, 594 and 636 proteins were identified in PACG, POAG and cataract groups, respectively (n = 9 in each group). The inter-group comparison among all these groups showed that 246 proteins were identified in all the three groups. An average of 236 ± 42, 218 ± 40 and 214 ± 62 proteins from each AH sample of PACG, POAG and cataract, respectively, was identified. There were 53 proteins commonly found in all 9 PACG AH, 59 proteins in POAG AH and 42 proteins in 9 cataracts AH samples. In the individual analysis, there were 28 proteins found in all the samples analyzed representing the "constitutive AH proteome." Spectral counting analysis of 246 proteins identified in all three group types showed significant differences in protein abundance. In proteins unique to PACG AH, 7 proteins viz. ARHGEF12, APC2, WAS, PIK3CG, ITGB1, MSN and PFN1 out of 226 were found in "Regulation of Actin Cytoskeleton" pathway, whereas in POAG 5 out of 206 proteins viz. ADCY2, ITPR1, MAPK3, MAP3K2 and TUBB1 were found in "Gap Junction" pathway. CONCLUSIONS: A qualitative as well as a quantitative comparison of proteomes of AH from PACG, POAG and age-related cataract eyes showed significant differences, thus providing clues to the disease pathophysiology.

Tear inflammatory mediators and protein in eyes of post allogenic hematopoeitic stem cell transplant patients.

AIM: To analyze tear cytokines levels and their correlation to ocular surface parameters in allogenic hematopoietic stem cell transplants (allo-HSCT) patients. METHODS: Prospective longitudinal study of allo-HSCT patients and controls for ocular surface evaluation (OSDI, TBUT, Schirmer's test, staining scores), tear biochemical analysis for protein, cytokines [IL-10, IL-12, IL-2, IL-4, IL-6, IL-17, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, VEGF], MMPs [MMP 2, 9, 7, 13, 10 and chemokine (IL-8)], & VEGF on three consecutive follow up visits (at three monthly interval) was done. RESULTS: Of 24 post allo-HSCT patients (19 males, 5 females) & 12 controls (mean age 34.3 + 5.8 years) enrolled, 20 patients [mean age 33.4 + 7.77 years; mean time of recruitment of 5.2 + 2.12 months following alloHSCT] who completed three consecutive follow up visits were included for analysis. Ocular GVHD (oGVHD) was seen in 8 patients (33.3%). Tears biochemical analysis showed elevated levels of interferon γ, IL 6, IL 8, IL 10, IL 12AP70, IL 17A, MMP 9 and VEGF in oGVHD eyes as compared to non-oGVHD & control eyes. Non-oGVHD eyes showed elevated tear MMP 7 and MMP 9 as compared to healthy controls. Tear protein levels were significantly decreased in oGVHD eyes and were equivocal in nonGVHD and control eyes. TBUT and ocular staining scores to correlate best with tear interleukins and MMPs. CONCLUSION: Evaluation of levels of tear VEGF, total protein & MMP 9 can be of significance in identifying oGVHD in post alloHSCT patients.

Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Role of CYP1B1, p.E229K and p.R368H mutations among 120 families with sporadic juvenile onset open-angle glaucoma.

BACKGROUND: To determine the frequency of CYP1B1 p.E229K and p.R368H, gene mutations in a cohort of sporadic juvenile onset open-angle glaucoma (JOAG) patients and to evaluate their genotype/phenotype correlation. METHODS: Unrelated JOAG patients whose first-degree relatives had been examined and found to be unaffected were included in the study. The patients and their parents were screened for p.E229K and p.R368H mutations. The phenotypic characteristics were compared between probands carrying the mutations and those who did not carry these mutations. RESULTS: Out of 120 JOAG patients included in the study, the p.E229K mutation was seen in 9 probands (7.5%) and p.R368H in 7 (5.8%). The average age of onset of the disease (p = 0.3) and the highest untreated IOP (p = 0.4) among those carrying mutations was not significantly different from those who did not have these mutations. The proportion of probands with angle dysgenesis among those with p.E229K and p.R368H mutations was 70% (11 out of 16) in comparison to 65% (67 out of 104) of those who did not harbour these mutations (p = 0.56). Similarly, the probands with moderate to high myopia among those with p.E229K and p.R368H mutations was 20% (3 out of 16) in comparison to 18% (18 out of 104) of those who did not harbour these mutations (p = 0.59). CONCLUSION: The frequency of p.E229K and p.R368H mutations of the CYP1B1 gene is low even among sporadic JOAG patients. Moreover, there is no clinical correlation between the presence of these mutations and disease severity.