Detection of red cell alloantibody prior to overflow phenomenon in a case of warm AIHA secondary to Chronic Lymphocytic Leukemia and Myasthenia Gravis: A case report.

INTRODUCTION: Defects in the lymphoid system have been linked to immune dysregulation, which might explain why lymphoid neoplasms and immunological disorders tend to occur concurrently. Chronic Lymphocytic Leukemia (CLL), characterised by the accumulation of dysfunctional lymphocytes, is associated with autoimmune cytopenias such as autoimmune haemolytic anaemia (AIHA). Detection of underlying alloantibody in warm AIHA, is challenging for any transfusion medicine specialist. This report highlights the significance of overflow phenomenon in detection of alloantibody in a case of warm AIHA secondary to CLL and myasthenia gravis. CASE REPORT: A 56-year-old male with a history of myasthenia gravis and thymoma progressed to B-cell CLL presented with severe anaemia and thrombocytopenia leading to multiple red blood cell (RBC) transfusions in the last two months. Clinical profile and laboratory workup suggested features of AIHA, and subsequent immunohaematological workup hinted towards an impending overflow phenomenon due to differential reactivity pattern observed between serum and eluate with antibody screen/identification panel. The eluate was pan-reactive with an antibody screen/ identification panel, while the serum showed a discrete anti-C alloantibody pattern. A compatible and antigen-negative RBC unit was successfully transfused, followed by medical management. DISCUSSION: The overflow phenomenon in AIHA depends on antibody titre and its affinity for RBC antigens. In the index case, the impending 'overflow or spillover' of autoantibodies into the patient's serum allowed us to detect underlying alloantibody without performing allogeneic adsorption and transfuse antigen-negative and crossmatch compatible PRBC unit. CONCLUSION: This case emphasises the significance of understanding the overflow phenomenon in AIHA as it can guide a transfusion medicine specialist in the early detection and identification of underlying alloantibodies, which is crucial for appropriate transfusion management in AIHA. However, early presentation and timely workup, along with a high level of suspicion, is crucial to identify this phenomenon.

Clinical Post-operative Bleeding During Minor Oral Surgical Procedure and In Vitro Platelet Aggregation in Patients on Aspirin Therapy: Are they Coherent?

AIM: The risk of excessive bleeding prompts physicians to discontinue aspirin in patients on low-dose, long-term therapy which in turn puts them at the risk from adverse cardiovascular and thrombotic events. Effect of low-dose aspirin therapy on platelet function was assessed using platelet aggregation method. The aim was to correlate the laboratory platelet function with cutaneous and clinical oral bleeding time (BT). MATERIALS AND METHODS: One hundred one patients were enrolled in this prospective trial and were allocated into two groups. Interventional or test group consisted of patients who were on aspirin therapy (75 mg/100 mg) for primary or secondary prevention of angina, myocardial infarction and stroke. Minor oral surgical procedure was performed in this group without discontinuing aspirin therapy. Control group consisted of healthy patients (under no medication) undergoing minor oral surgical procedure. Cutaneous and clinical oral BT were recorded in both the groups. Venous blood sample was drawn, and percentage platelet aggregation function was analysed using adenosine diphosphate (ADP) and arachidonic acid (AA) reagents. The percentage of platelet aggregation was then correlated with cutaneous and clinical oral BT. RESULTS: A significant decrease in percentage platelet aggregation using ADP (aspirin-74.7 21.39; control-89.2 13.70) and AA (aspirin-47.6 23.11; control-82.3 20.17) was observed. However, there were no significant difference in mean cutaneous BT (aspirin-1.5 0.65 min; control-1.6 0.71 min) and clinical oral BT (aspirin-5.0 2.48 min; control-4.8 2.60 min) in aspirin and control groups. CONCLUSION: Majority of the minor oral surgical procedures can be carried out safely without discontinuing aspirin in patients on low-dose long-term therapy. This is possible because despite significant platelet aggregation evident in laboratory evaluation there is lack of its clinical corroboration owing to aspirin resistance. CLINICAL TRIAL REGISTRATION: CTRI/2018/02/012055.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

BACKGROUND: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. METHODS: Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. RESULTS: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). CONCLUSION: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

Andrographolide engineered gold nanoparticle to overcome drug resistant visceral leishmaniasis.

Visceral leishmaniasis (VL) is World Health Organization designated most serious leishmaniasis with an annual mortality rate of 50,000. Even after country specific eradication programs, the disease continues to multiply with added complexities like resistance development, drug hypersensitivity and associated infections. Newer therapeutic interventions are urgently warranted to control the spread. Present study aims to arrive at terpenoid andrographolide engineered gold nanoparticle (AGAunps) facile synthesis and its efficacy evaluations against wild and drug resistant VL strains for the first time. Molecular bio-organic conjugation of AGAunp was confirmed in FT-IR and EDAX studies. Nano-gold plasmon response was recorded at 543 nm and the average size in TEM was 14 nm. SAED pattern and XRD observations proved fcc crystalline structure of nano-gold. AGAunp recorded spherical geometry in AFM and TEM. PDI value of 0.137 revealed the monodisperse nature of the nano-scale population. AGAunp exhibited strong antileishmanicidal effect both against wild type (IC50 19 ± 1.7 µM) and sodium stibogluconate (IC50 55 ± 7.3 µM)/paromomycin (IC50 41 ± 6 µM) resistant strains. Complete macrophage uptake AGAunp's occured within two hours exposure. AGAunp macrophage cytotoxicity was significantly lower as compared to Amphotericin-B. Low toxic Andrographolide engineered gold nanoparticle emerged as promising alternatives in the control of wild and drug resistant VL.

Adjunct therapeutic plasma exchange for anti-N-methyl-D-aspartate receptor antibody encephalitis: a case report and review of literature.

Encephalitis associated with autoantibodies directed against the N-methyl-D-aspartate receptor (NMDAR) is usually a paraneoplastic syndrome that presents in young females with ovarian teratomas. We report a case of a previously healthy 14-year-old girl with sudden-onset paranoia, hallucinations, hyperactivity, increased speech, decreased sleep, seizures, and violent behavior deteriorating to catatonia. Her cerebrospinal fluid tested positive for anti-NMDAR antibodies. She was treated with five sessions of therapeutic plasma exchange (TPE) after having failed therapy with antibiotics, intravenous steroids, intravenous immunoglobulin (IVIG), one dose of rituximab, and seven sessions of electroconvulsive therapy (ECT). The American Society for Apheresis assigns a Category III (Grade 2C) recommendation for TPE in paraneoplastic neurologic syndromes; however, apheresis specifically for anti-NMDAR encephalitis has not been well studied. Literature review revealed two case reports describing outstanding improvement in patients with anti-NMDAR encephalitis following TPE. We report no improvement in our patient's symptoms after plasma exchange and discuss possible reasons for why it failed along with review of the literature.

Investigation of the cause of death in a gene-therapy trial.

We present a case of disseminated histoplasmosis, complicated by retroperitoneal bleeding and leading to death, in a patient who was receiving systemic immunosuppressive therapy for rheumatoid arthritis and who was enrolled in a gene-therapy trial. This trial was designed to evaluate intraarticular delivery of a tumor necrosis factor alpha (TNF-alpha) antagonist, through an adeno-associated virus (AAV) type 2 delivery system, for inflammatory arthritis. The patient's receipt of concurrent anti-TNF-alpha therapy and other immunosuppressive therapy while she was living in an area where histoplasmosis was endemic was thought to be the most likely explanation for the infection; the evidence presented suggests that this fatal infection was unlikely to have been related to exposure to the agent administered in the gene-therapy trial. This case reinforces the importance of considering infectious complications, such as those from endemic mycoses, in patients receiving treatment with a TNF-alpha antagonist and the importance of having a well-designed monitoring plan when subjects in a research study become ill. (ClinicalTrials.gov number, NCT00126724.)

Renal medullary carcinoma: rhabdoid features and the absence of INI1 expression as markers of aggressive behavior.

Renal medullary carcinoma is a rare, well-recognized highly aggressive tumor of varied histopathology, which occurs in young patients with sickle cell trait or disease. Rhabdoid elements, occasionally seen in high-grade renal tumors including renal medullary carcinoma, possibly represent a pathologic marker of aggressive behavior. INI1 (hSNF5/SMARCB1/BAF47) is a highly conserved factor in the ATP-dependent chromatin-modifying complex. Loss of this factor in mice results in aggressive rhabdoid tumors or lymphomas. In humans, the loss of INI1 expression has been reported in pediatric renal rhabdoid tumors, central nervous system atypical teratoid/rhabdoid tumors and epithelioid sarcomas, a possible primary soft tissue rhabdoid tumor. This study compares five renal medullary carcinomas with 10 high-grade renal cell carcinomas (five with rhabdoid features), two urothelial carcinomas and two pediatric renal rhabdoid tumors. All five renal medullary carcinomas, irrespective of histopathology, showed complete loss of INI1 expression similar to that seen in pediatric renal rhabdoid tumors. In contrast, all renal cell carcinomas or urothelial carcinomas, including those with histological rhabdoid features, expressed INI1. Clinically, all five of the patients with renal medullary carcinoma and the two patients with rhabdoid tumors presented with extra-renal metastases at the time of diagnosis. This study demonstrates that renal medullary carcinoma and renal rhabdoid tumor share a common molecular/genetic alteration, which is closely linked to their aggressive biological behavior. However, the absence of INI1 expression is not necessarily predictive of rhabdoid histopathology but remains associated with aggressive behavior in renal medullary carcinoma.

ProFace: a server for the analysis of the physicochemical features of protein-protein interfaces.

BACKGROUND: Molecular recognition is all pervasive in biology. Protein molecules are involved in enzyme regulation, immune response, signal transduction, oligomer assembly, etc. Delineation of physical and chemical features of the interface formed by protein-protein association would allow us to better understand protein interaction networks on one hand, and to design molecules that can engage a given interface and thereby control protein function on the other hand. RESULTS: ProFace is a suite of programs that uses a file, containing atomic coordinates of a multi-chain molecule, as input and analyzes the interface between any two or more subunits. The interface residues are shown segregated into spatial patches (if such a clustering is possible based on an input threshold distance) and/or core and rim regions. A number of physicochemical parameters defining the interface is tabulated. Among the different output files, one contains the list of interacting residues across the interface. Results can be used to infer if a particular interface belongs to a homodimeric molecule. CONCLUSION: A web-server, ProFace (available at http://www.boseinst.ernet.in/resources/bioinfo/stag.html) has been developed for dissecting protein-protein interfaces and deriving various physicochemical parameters.