Publications by authors named "Rahul Krishnan"

32 Publications

Deep learning and the future of the Model for End-Stage Liver Disease-sodium score.

Liver Transpl 2022 Apr 19. Epub 2022 Apr 19.

Ajmera Transplant Program, University Health Network, Toronto, Ontario, Canada.

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http://dx.doi.org/10.1002/lt.26485DOI Listing
April 2022

Beta glucan induced immune priming protects against nervous necrosis virus infection in sevenband grouper.

Fish Shellfish Immunol 2022 Feb 10;121:163-171. Epub 2022 Jan 10.

Department of Aqualife Medicine, Chonnam National University, Yeosu, 59629, Republic of Korea. Electronic address:

In the present study, we studied the effect of β-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of β-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1β production were reduced in β-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the β-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with β-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that β-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.
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http://dx.doi.org/10.1016/j.fsi.2022.01.005DOI Listing
February 2022

Altered expression of immune factors in sevenband grouper, Hyporthodus septemfasciatus following nervous necrosis virus challenge at optimal and suboptimal temperatures.

Fish Shellfish Immunol 2021 Dec 23;119:442-451. Epub 2021 Oct 23.

Department of Aqualife Medicine, Chonnam National University, Yeosu, Republic of Korea. Electronic address:

The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1β and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 10 TCID/100μL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.
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http://dx.doi.org/10.1016/j.fsi.2021.10.033DOI Listing
December 2021

Gargle lavage & saliva: Feasible & cheaper alternatives to nasal & throat swabs for diagnosis of COVID-19.

Indian J Med Res 2021 05;153(5&6):665-670

Department of Medicine, All India Institute of Medical Sciences, New Delhi, India.

Background & Objectives: In the present scenario, the most common sample for diagnosis of COVID-19 by reverse transcription polymerase chain reaction (RT-PCR) is nasal and throat swab (NTS). Other sampling options such as gargle lavage have found limited application in clinical use mostly because of unavailability of an appropriate gargling liquid. This study was conducted to assess the stability of SARS-CoV-2 RNA in normal saline at 4°C that can serve as a gargling liquid as well as a transport medium. The study also looked at the agreement between NTS and gargle lavage/saliva for the detection of SARS-CoV-2.

Methods: In 29 consecutive real-time RT-PCR (rRT-PCR) positive COVID-19 patients, paired NTS, gargle and saliva samples were taken. Samples were processed by rRT-PCR for the detection of SARS-CoV-2 RNA. To assess the SARS-CoV-2 RNA stability in normal saline, gargle lavage specimens were divided into two aliquots; one subset of the specimen was run within 4-6 h along with the routine samples (NTS and saliva) and the other subset was stored at 4°C and processed after 24-30 h. Agreement between cycle threshold (Ct) values from both the runs was compared using Bland-Altman (BA) analysis.

Results: The positivity rates of rRT-PCR in NTS, saliva and gargle lavage samples were 82.7 (24/29), 79.3 (23/29) and 86.2 per cent (25/29), respectively. BA plot showed a good agreement between the Ct values of fresh and stored gargle samples, stipulating that there were no significant differences in the approximate viral load levels between the fresh and stored gargle lavage samples (bias: E gene -0.64, N gene -0.51, ORF gene -0.19).

Interpretation & Conclusions: Our study results show stability of SARS-CoV-2 RNA in the gargle samples collected using normal saline up to 24-30 h. Gargle lavage and saliva specimen collection are cost-effective and acceptable methods of sampling for the detection of SARS-CoV-2 RNA by rRT-PCR. These simplified, inexpensive and acceptable methods of specimen collection would reduce the cost and workload on healthcare workers for sample collection.
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http://dx.doi.org/10.4103/ijmr.IJMR_4209_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8555589PMC
May 2021

An Investigation Using Specific Absorption Rate Analysis to Diagnose Early- stage Breast Tumor using UWB Antenna.

Curr Med Imaging 2021 ;17(12):1425-1431

Department of Biomedical Engineering, Rajalakshmi Engineering College, Chennai, India.

Aims: This work was focused on the detection of early-stage breast tumors and their location.

Background: The most frequently seen disease progression and mortality in women's lives is Breast Cancer. Because of breast cancer/tumors, the risk of mortality for women has risen exponentially. From the 2020 deadline for breast cancer, women who died from carcinoma were 123.8 cases per lac women between 2006-2010. This problem is also overcome by the early identification of the tumor using different detection procedures like X-ray mammography, computerized tomography, ultrasound imaging technique, Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), microwave imaging. Researchers carried out multiple studies in these areas.

Objective: To detect early-stage breast tumors and their location using SAR analysis.

Methods: The major dielectrical difference between cancerous breast tissues and normal tissues in this technique is the microwave frequency range. The term Specific Absorption Rate (SAR) describes the amount of energy which is absorbed (W/kg) in the breast tissue. This segment illustrates the usefulness to diagnose the tumor position in the breast by means of maximum SAR value coordinates. Changes in breast and tumor size are important for the risk of diagnosis. The power absorbed in connecting with a normal breast and a tumor breast is measured and equivalent for different breast masses. The maximum SAR is also analyzed at distinct tumor locations at various frequency ranges.

Results: It is observed that max SAR coordinates are very close to the actual tumor location. So, the maximal value of SAR coordinates indicates the existence of a tumor in the breast phantom.

Conclusion: The simulated data above strongly suggests that the Max SAR values were higher in the breast phantom with tumor as compared to the breast without tumor. With different tumor radius (3 mm and 5 mm) analyzed with different resonant frequencies like 3GHz, 4GHz and 5GHz at the actual tumor location of (0, 0, 35). Even though a model representing the real properties of breast tissue is required to assess the validation of any imaging process, so the real-time development of an equivalent breast phantom and its execution is needed.
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http://dx.doi.org/10.2174/1573405617666210629121013DOI Listing
January 2022

Congenital Colonic Stenosis Manifested after Foreign-Body (Button Battery) Ingestion in a Child.

J Indian Assoc Pediatr Surg 2021 Jan-Feb;26(1):65-66. Epub 2021 Jan 11.

Department of General Surgery, Baptist Hospital, Bengaluru, Karnataka, India.

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http://dx.doi.org/10.4103/jiaps.JIAPS_95_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074825PMC
January 2021

Dose-dependent co-infection of Argulus sp. and Aeromonas hydrophila in goldfish (Carassius auratus) modulates innate immune response and antioxidative stress enzymes.

Fish Shellfish Immunol 2021 Jul 1;114:199-206. Epub 2021 May 1.

Aquatic Environment and Health Management Division, ICAR- Central Institute of FisheriesEducation, Mumbai, 400061, Maharashtra, India. Electronic address:

Co-infection with parasites and bacteria is of frequent occurrence in aquaculture, leads to growth impedance otherwise mortality in fish depending on the varying degree of a load of primary pathogen either parasite or bacteria. The mechanistic regulation of immune response during co-infection in fish has merely documented. The aim of this study was to determine the impact of co-infection with Aeromonas hydrophila at three exposure doses of Argulus sp. on the innate immune responses and antioxidative stress enzymes of goldfish (Carassius auratus). The experimental fish were randomly distributed into eight treatment groups viz. T1 (control group without Argulus and A. hydrophila infection), T2 (fish exposed to a sub-lethal dose of A. hydrophila), T3 (low Argulus-infested fish), T4 (T3 + sub-lethal dose of A. hydrophila), T5 (moderate Argulus-infested fish), T6 (T5 + sub-lethal dose of A. hydrophila), T7 (high Argulus-infested fish) and T8 (T7+ sub-lethal dose of A. hydrophila) in duplicates. After distributing experimental fish into their respective treatment group, A. hydrophila was injected to T2, T4, T6 and T8. After the bacterial challenge, four fish from each experimental group were randomly sampled on 24, 72, and 168 h and subjected to the hematological, innate immune parameters and enzymatic analysis. In the co-infection group T8, a high degree of enhanced pathogenicity of A. hydrophila was noticed with increased mortalities (84.2%) in comparison to other groups. The current study shows a declining pattern in RBC, PCV and Hb values with the degree of parasite infestation without co-infection groups. Moreover, in the T8 group, exposure of a sub-lethal dose of bacteria resulted in a drastic reduction of the recorded parameters. Furthermore, a decreased value for WBC, monocyte and neutrophil was found in higher parasite group co-infected with a sub-lethal dose of bacteria relative to other co-infected groups during the experimental period. Also, a decrease in innate immune parameters and antioxidative stress enzymes were observed in the T8 group compared to T7 and T2 groups throughout the trial period. These findings indicate that a rise in the dose of Argulus infection improves A. hydrophila colonization in goldfish and contributes to suppression of the innate immune system and increased mortality.
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http://dx.doi.org/10.1016/j.fsi.2021.04.026DOI Listing
July 2021

Proteasome subunit beta type-8 from sevenband grouper negatively regulates cytokine responses by interfering NF-κB signaling upon nervous necrosis viral infection.

Fish Shellfish Immunol 2021 Jun 13;113:118-124. Epub 2021 Apr 13.

Department of Aqualife Medicine, Chonnam National University, Yeosu, Republic of Korea. Electronic address:

During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome β-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1β, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.
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http://dx.doi.org/10.1016/j.fsi.2021.04.004DOI Listing
June 2021

Concordance Between Obstetric Anatomic Ultrasound and Fetal Echocardiography in Detecting Congenital Heart Disease in High-risk Pregnancies.

J Ultrasound Med 2021 Oct 10;40(10):2105-2112. Epub 2020 Dec 10.

Department of Obstetrics and Gynecology, University of Virginia, Charlottesville, Virginia, USA.

Objectives: To evaluate the concordance between second-trimester anatomic ultrasound and fetal echocardiography in detecting minor and critical congenital heart disease in pregnancies meeting American Heart Association criteria.

Methods: We conducted a retrospective cohort study of pregnancies in which a second-trimester fetal anatomic ultrasound examination (18-26 weeks) and fetal echocardiography were performed between 2012 and 2018 at our institution based on American Heart Association recommendations. Anatomic ultrasound studies were interpreted by maternal-fetal medicine specialists and fetal echocardiographic studies by pediatric cardiologists. Our primary outcome was the proportion of critical congenital heart disease (CCHD) cases not detected by anatomic ultrasound but detected by fetal echocardiography. The secondary outcome was the proportion of total congenital heart disease cases missed by anatomic ultrasound but detected by fetal echocardiography. Neonatal medical records were reviewed for all pregnancies when obtained and available.

Results: Overall, 722 studies met inclusion criteria. Anatomic ultrasound and fetal echocardiography were in agreement in detecting cardiac abnormalities in 681(96.1%) studies (κ = 0.803; P < .001). The most common diagnosis not identified by anatomic ultrasound was a ventricular septal defect, accounting for 9 of 12 (75%) missed congenital heart defects. Of 664 studies with normal cardiac findings on the anatomic ultrasound examinations, no additional instances of CCHD were detected by fetal echocardiography. No unanticipated instances of CCHD were diagnosed postnatally.

Conclusions: With current American Heart Association screening guidelines, automatic fetal echocardiography in the setting of normal detailed anatomic ultrasound findings provided limited benefit in detecting congenital heart defects that would warrant immediate postnatal interventions. More selective use of automatic fetal echocardiography in at-risk pregnancies should be explored.
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http://dx.doi.org/10.1002/jum.15592DOI Listing
October 2021

(Phylum Dicyemida: Dicyemidae) isolated from Korean common octopus in Korea.

J Vet Sci 2020 Nov;21(6):e86

Department of Marine Life Science & Marine Science Institute, Jeju National University, Jeju 63243, Korea.

Background: Dicyemids are parasites found in the renal sac of cephalopods. The first species of dicyemid was found from kidneys of the Korean common octopus .

Objectives: This study aimed to identify the dicyemid and investigate the effect on renal sac of host.

Methods: In this study, we compared the morphological characteristics of isolate to dicyemids (, , and ) reported from in Japan. We compared the 18S ribosomal RNA (rDNA) and cytochrome c oxidase subunit I (COI) sequences of isolate to the sequences of and . The infected octopuses renal tissues were histologically compared with the tissues of uninfected individuals.

Results: The morphological characteristic of this isolated species corresponds to . The sequences similarities of 18S rDNA and COI gene of isolate are 99.7% and 98.1% with . We observed morphological changes in the epithelia folds of kidney at the dicyemids attached areas.

Conclusions: The present study identified the isolate as and this is the first report of dicyemid species from Republic of Korea. Further studies on the effects of dicyemids on growth and health status of cephalopods will be needed.
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http://dx.doi.org/10.4142/jvs.2020.21.e86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710466PMC
November 2020

Detection of New Delhi Metallo-β-lactamase 1 and Cephalosporin Resistance Genes Among Carbapenem-Resistant Enterobacteriaceae in Water Bodies Adjacent to Hospitals in India.

Curr Microbiol 2020 Oct 8;77(10):2886-2895. Epub 2020 Jul 8.

Department of Aquatic Animal Health Management, Kerala University of Fisheries and Ocean Studies, Kochi, Kerala, India.

The prevalence of carbapenem resistance among bacterial isolates from selected water bodies receiving hospital effluents and adjoining aquaculture farms in Kerala, India, was studied. Klebsiella pneumoniae followed by Escherichia coli, Klebsiella oxytoca, Enterobacter aerogenes and Acinetobacter baumannii were the predominant isolates. Antibiotic sensitivity of these isolates was determined by Kirby-Bauer disc diffusion method. Nearly 60% of the Enterobacteriaceae isolates screened were multidrug resistant of which 16.6% were carbapenem resistant. The carbapenem-resistant Enterobacteriaceae were further screened for the presence of New Delhi metallo β-lactamase-1 and cephalosporin resistance encoding genes. All NDM-1 isolates were highly resistant to carbapenem, cephalosporin, aminoglycosides, quinolones, tetracycline, and sulphonamides. K. pneumoniae harboring bla gene and E. coli isolates with bla and bla genes were detected in hospital discharge points. In aquaculture farms too, carbapenem-resistant K. pneumoniae with bla gene and E. coli isolates with bla were observed, although there was no use of antibiotics in these farms. However, other carbapenemase genes such as bla, bla, bla and bla were not detected in any of these isolates. The results suggest the increased prevalence of carbapenem-resistant Enterobacteriaceae in the water bodies receiving hospital effluent and its dissemination to adjacent aquaculture farms, posing a serious threat to public health.
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http://dx.doi.org/10.1007/s00284-020-02107-yDOI Listing
October 2020

Early viral uptake and host-associated immune response in the tissues of seven-band grouper following a bath challenge with nervous necrosis virus.

Fish Shellfish Immunol 2020 Aug 18;103:454-463. Epub 2020 May 18.

Department of Aqualife Medicine, Chonnam National University, Yeosu, Republic of Korea. Electronic address:

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.
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http://dx.doi.org/10.1016/j.fsi.2020.05.012DOI Listing
August 2020

β-catenin and PD-L1 expression in mismatch repair deficient endometrial carcinomas.

Int J Gynecol Cancer 2020 07 5;30(7):993-999. Epub 2020 May 5.

Obstetrics & Gynecology, University of Virginia Health System, Charlottesville, Virginia, USA

Introduction: Predictors of non-response in mismatch repair deficiency cancers are poorly understood. Upregulation of the canonical Wnt pathway has been associated with decreased immune cell infiltration in many cancer types. The relationship between Wnt/β-catenin pathway activation and the programmed death-ligand 1 axis in endometrial cancer remains poorly characterized. This study evaluates β-catenin expression in a well characterized cohort of endometrial cancers by mismatch repair status and programmed death-ligand 1 expression.

Methods: Whole sections of formalin-fixed, paraffin embedded tissue from 23 Lynch syndrome-associated carcinomas, 20 mutL homolog-1 (MLH1) promoter hypermethylated carcinomas, and 19 mismatch repair intact carcinomas were evaluated. Immunohistochemistry staining for β-catenin and programmed death-ligand 1 was performed on all cases. Programmed death-ligand 1 expression was scored in both the tumor and the peri-tumoral immune compartment. Tumor staining was classified as positive when membranous (programmed death-ligand 1) staining was present in ≥1% of tumor cells. Immune stromal staining was scored as positive when ≥5% of peritumoral and intratumoral immune cells (including lymphocytes and macrophages) showed reactivity.

Results: Six tumors (6/62, 9.7%) demonstrated nuclear expression of β-catenin (4 were Lynch syndrome-associated, 1 was MLH1 methylated, 1 was mismatch repair intact). The majority of tumors with nuclear β-catenin expression demonstrated concomitant tumoral programmed death-ligand 1 expression (5/6, 83.3%) and were more likely to demonstrate tumoral programmed death-ligand 1 expression compared to tumors without nuclear β-catenin expression (83.3% vs 39.3%, p=0.04). Both tumoral and immune cell expression of programmed death-ligand 1 was statistically significantly associated with mismatch repair deficient tumors.

Discussion: Tumors demonstrating nuclear β-catenin expression were more likely to express tumoral programmed death-ligand 1 staining than tumors without nuclear β-catenin expression. Nuclear β-catenin expression could be a potential predictive biomarker for non-response to immune checkpoint inhibition in mismatch repair deficient tumors. Nuclear β-catenin expression status should be considered as a translational endpoint in future clinical trials of immune checkpoint inhibition in endometrial cancer.
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http://dx.doi.org/10.1136/ijgc-2020-001239DOI Listing
July 2020

An Enteropathy-like Indolent NK-Cell Proliferation Presenting in the Female Genital Tract.

Am J Surg Pathol 2020 04;44(4):561-565

Pathology.

Natural killer (NK) cell enteropathy is a lymphoproliferative disorder, initially described by Mansoor and colleagues, that presents in the gastrointestinal tract, and is often mistaken for extranodal NK/T-cell lymphoma on first assessment. This population of cells in this process have an NK-cell phenotype (CD3, CD56, CD2, CD7), lacks evidence of Epstein-Barr virus infection, has germline rearrangement of the T-cell receptor, and a very indolent clinical course. Indeed, many of such patients had been originally diagnosed as having an NK/T-cell lymphoma, and subsequently received chemotherapy. We report a unique case where an indolent lymphoproliferative disorder with features that resemble NK-cell enteropathy is encountered for the first time outside the gastrointestinal tract, specifically in the female genitourinary tract. We provide morphologic, immunophenotypic, and molecular documentation of such, in association with a completely indolent clinical behavior of this type of process.
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http://dx.doi.org/10.1097/PAS.0000000000001387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071997PMC
April 2020

Functional characterization of seven-band grouper immunoglobulin like cell adhesion molecule, Nectin4 as a cellular receptor for nervous necrosis virus.

Fish Shellfish Immunol 2019 Oct 9;93:720-725. Epub 2019 Aug 9.

Department of Aqualife Medicine, Chonnam National University, Yeosu, Republic of Korea. Electronic address:

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.
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http://dx.doi.org/10.1016/j.fsi.2019.08.019DOI Listing
October 2019

Interferon-regulatory factors, IRF3 and IRF7 in Asian seabass, Lates calcarifer: Characterization, ontogeny and transcriptional modulation upon challenge with nervous necrosis virus.

Fish Shellfish Immunol 2019 Jun 30;89:468-476. Epub 2019 Mar 30.

Aquatic Environment and Health Management Division, ICAR- Central Institute of Fisheries Education, Mumbai, 400061, India.

Interferon regulatory factor (IRF) 3 and IRF7 are key regulators of type I interferon (IFN) gene expression for the antiviral immune response. In the present study, interferon regulatory factor 3 and 7 from Asian seabass, namely AsIRF3 and AsIRF7 were cloned and characterized. The full-length cDNA sequence of IRF3 and IRF7 consisted of 2965 and 2343 bp respectively. AsIRF3 and AsIRF7 were true orthologes of vertebrate IRF3/7 and showed similar domain organization, with an N-terminal DBD which consisted five tryptophan residues in IRF3 and four in IRF7, a C-terminal IRF3 domain and a serine rich region. Both IRF3 and 7 constitutively expressed during the ontogenesis and in all tissues of healthy fish. The expression of both genes was up-regulated following NNV challenge with obvious transcript abundance in brain heart and kidney. Ectopic expression of AsIRF3 and AsIRF7 displayed activation of ISRE/NF-κB promoters and modulation of interferon, ISGs and pro-inflammatory cytokine gene expression. These observations indicated that IRF3 and IRF7 play an important role in Asian seabass's antiviral defense and the RIG-IRF-IFN axis is conserved in the species.
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http://dx.doi.org/10.1016/j.fsi.2019.03.073DOI Listing
June 2019

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

J Virol Methods 2019 02 8;264:1-10. Epub 2018 Nov 8.

Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu, 59626, Republic of Korea. Electronic address:

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.
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http://dx.doi.org/10.1016/j.jviromet.2018.11.003DOI Listing
February 2019

Representation Learning Approaches to Detect False Arrhythmia Alarms from ECG Dynamics.

Proc Mach Learn Res 2018 Aug;85:571-586

Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA.

The high rate of intensive care unit false arrhythmia alarms can lead to disruption of care and slow response time due to desensitization of clinical staff. We study the use of machine learning models to detect false ventricular tachycardia (v-tach) alarms using ECG waveform recordings. We propose using a Supervised Denoising Autoencoder (SDAE) to detect false alarms using a low-dimensional representation of ECG dynamics learned by minimizing a combined reconstruction and classification loss. We evaluate our algorithms on the PhysioNet Challenge 2015 dataset, containing over 500 records (over 300 training and 200 testing) with v-tach alarms. Our results indicate that using the SDAE on Fast Fourier Transformed (FFT) ECG at a beat-by-beat level outperforms several competitive baselines on the task of v-tach false alarm classification. We show that it is important to exploit the underlying known physiological structure using beat-by-beat frequency distribution from multiple cardiac cycles of the ECG waveforms to obtain competitive results and improve over previous entries from the 2015 PhysioNet Challenge.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853621PMC
August 2018

Preliminary investigations on the role of Drp-1 dependent mitochondrial fission in attenuating RLR downstream signaling during nervous necrosis virus infection.

Fish Shellfish Immunol 2018 Sep 4;80:618-623. Epub 2018 Jul 4.

Aquatic Environment and Health Management Division, ICAR- Central Institute of Fisheries Education, Mumbai, India.

Member of the dynamin family of large GTPases, dynamin-related protein 1 (Drp1) dependent mitochondrial fission is an intricate process regulating both cellular and organ dynamics. Present study shows that NNV perturbs mitochondrial dynamics by promoting Drp-1 dependent mitochondrial fission, which attenuates MAVS mediated downstream signaling. NNV infected SISS cells revealed induction in Drp1 expression and subsequent translocation into mitochondria. The level of MAVS expression was up-regulated over a period of 24 hpi and declined with the progression of NNV infection at 48 and 72 hpi confirmed by western blot and mRNA transcript analysis. Drp-1 displayed its association with fragmented mitochondria and the transcript abundance was significant post infection along with Mff. Expression levels of IRF-3 IFN-1 and Mx followed a similar pattern with abundant expression at 48 hpi and diminished expression during the further period. Importantly, silencing of Drp1 caused significant elevation in the RLR downstream molecules and reduction in viral RNA expression. These results suggest that NNV-induced mitochondrial fission serve to attenuate host RLR signaling. This provides an illustration of host-pathogen interaction in which the virus evades innate immunity by enhancing mitochondrial fission and perturbs MAVS, and the downstream molecules.
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http://dx.doi.org/10.1016/j.fsi.2018.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111691PMC
September 2018

Antiviral activity of transiently expressed mitochondrial antiviral signaling adapter, MAVS orthologue from Asian seabass.

Fish Shellfish Immunol 2018 May 3;76:183-186. Epub 2018 Mar 3.

Aquatic Environment and Health Management Division, ICAR- Central Institute of Fisheries Education, Mumbai, India. Electronic address:

The innate immune signaling adapter, Mitochondrial antiviral signaling protein (MAVS) coordinates the signals received from two independent RLRs (RIG-1 and MDA5) to induce IFN & interferon stimulatory genes (ISGs). In the present study, we report identification of an orthologue of MAVS from Lates calcarifer (LcMAVS) and its functional role in piscine RLR signaling. The LcMAVS-cDNA was cloned into pcDNA and transfected into SISS cells. LcMAVS was detected to be a 61KDa protein in western blot. Confocal microscopy demonstrated the mitochondrial localization of LcMAVS. In addition, pcDNA-MAVS transfected cells were protected against Nervous Necrosis Virus (NNV) infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral transcript levels. Ectopic expression of LcMAVS resulted in activation of an ISRE-containing promoter (52 folds over control cells) as well as transcriptional expression of IRF-3, IFN-1 and IFN-inducible genes including Mx and ISG15 (p<0.05). These results suggest that LcMAVS is involved in the antiviral immunity as one of the adaptors in fish IFN-activation pathway.
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http://dx.doi.org/10.1016/j.fsi.2018.03.003DOI Listing
May 2018

Molecular cloning, characterization and expression profiling of galectin-9 gene from Labeo rohita (Hamilton, 1822).

Fish Shellfish Immunol 2018 May 15;76:287-292. Epub 2018 Mar 15.

Fish Genetics and Biotechnology Division, ICAR- Central Institute of Fisheries Education, Mumbai 61, India.

Galectin-9 is a b-galactoside-binding tandem repeat galectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the normal expression and immune response of Labeo rohita to pathogens, a tandem-repeat galectin-9 from Labeo rohita was identified and named LrGal-9. Its full-length cDNA was 1534 bp encoded 291 amino acids (35.12 KDa), shared the highest 81% identity with the galectin-9 of Danio rerio. LrGal-9 identified in this study lacked signal peptide and a transmembrane domain like galectin-9 members reported in other fishes. Quantitative PCR showed that LrGal-9 was lowly expressed in gill, muscle, heart, highly expressed in tested immune tissues (intestine, kidney, liver, spleen) in normal body. After Aeromonas hydrophila challenge, LrGal-9 was remarkably increased in all tested immune tissues in a time-dependent manner. These results suggest that LrGal-9 plays a role in innate immunity in Labeo rohita.
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http://dx.doi.org/10.1016/j.fsi.2018.02.037DOI Listing
May 2018

Molecular characterization, ontogeny and expression profiling of mitochondrial antiviral signaling adapter, MAVS from Asian seabass Lates calcarifer, Bloch (1790).

Dev Comp Immunol 2018 02 31;79:175-185. Epub 2017 Oct 31.

Aquatic Environment and Health Management Division, ICAR- Central Institute of Fisheries Education, Mumbai, India. Electronic address:

Mitochondrial antiviral signaling protein (MAVS), an innate immune signaling adapter coordinates the signals received from two independent cytosolic pathogen recognition receptors (RIG-1 and MDA5) to induce antiviral genes. In the present study the MAVS gene of Lates calcarifer (LcMAVS) was cloned and characterized. The complete cDNA sequence of LcMAVS was 3160 bp and encodes a poly peptide of 577 amino acids. Structural analysis of LcMAVS revealed an N-terminal CARD-like domain, central proline-rich domain and a C-terminal transmembrane domain. Phylogenetic analysis indicated that LcMAVS exhibited the closest relationship to P. olivaceous MAVS. LcMAVS was ubiquitously expressed in all tested tissues of healthy fish viz., brain, gill, heart, liver, spleen, kidney and intestine, with highest transcript level in spleen. The mRNA transcript level of LcMAVS in different developmental stages showed constitutive expression in all the stages tested suggesting the maternal transfer of the gene. Significant up regulation in MAVS expression was observed post nervous necrosis virus (NNV) challenge in vivo in all the selected tissues. Further, time course analysis showed that LcMAVS transcripts significantly increased in the brain and spleen tissues after NNV infection. These findings provide useful information for further elucidating the function of LcMAVS in antiviral innate immune response against NNV in Asian seabass.
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http://dx.doi.org/10.1016/j.dci.2017.10.019DOI Listing
February 2018

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials.

Methods Mol Biol 2017 ;1479:305-333

Department of Surgery, University of California Irvine, 333 City Blvd West, Suite 1600, Orange, CA, 92868, USA.

Transplantation of alginate-encapsulated islets has the potential to treat patients suffering from type I diabetes, a condition characterized by an autoimmune attack against insulin-secreting beta cells. However, there are multiple immunological challenges associated with this procedure, all of which must be adequately addressed prior to translation from trials in small animal and nonhuman primate models to human clinical trials. Principal threats to graft viability include immune-mediated destruction triggered by immunogenic alginate impurities, unfavorable polymer composition and surface characteristics, and release of membrane-permeable antigens, as well as damage associated molecular patterns (DAMPs) by the encapsulated islets themselves. The lack of standardization of significant parameters of bioencapsulation device design and manufacture (i.e., purification protocols, surface-modification grafting techniques, alginate composition modifications) between labs is yet another obstacle that must be overcome before a clinically effective and applicable protocol for encapsulating islets can be implemented. Nonetheless, substantial progress is being made, as is evident from prolonged graft survival times and improved protection from immune-mediated graft destruction reported by various research groups, but also with regard to discoveries of specific pathways involved in explaining observed outcomes. Progress in the latter is essential for a comprehensive understanding of the mechanisms responsible for the varying levels of immunogenicity of certain alginate devices. Successful translation of encapsulated islet transplantation from in vitro and animal model testing to human clinical trials hinges on application of this knowledge of the pathways and interactions which comprise immune-mediated rejection. Thus, this review not only focuses on the different factors contributing to provocation of the immune reaction by encapsulated islets, but also on the defining characteristics of the response itself.
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http://dx.doi.org/10.1007/978-1-4939-6364-5_24DOI Listing
December 2017

Developing a Rapid Algorithm to Enable Rapid Characterization of Alginate Microcapsules.

Cell Transplant 2017 05 10;26(5):765-772. Epub 2016 Oct 10.

The islets of Langerhans are endocrine tissue clusters that secrete hormones that regulate the body's glucose, carbohydrate, and fat metabolism, the most important of which is insulin, a hormone secreted by β-cells within the islets. In certain instances, a person's own immune system attacks and destroys them, leading to the development of type 1 diabetes (T1D), a life-long condition that needs daily insulin administration to maintain health and prolong survival. Islet transplantation is a surgical procedure that has demonstrated the ability to normalize blood sugar levels for up to a few years, but the need for chronic immunosuppression relegates it to a last resort that is often only used sparingly and in seriously ill patients. Islet microencapsulation is a biomedical innovation designed to protect islets from the immune system by coating them with a biocompatible polymer, and this new technology has demonstrated various degrees of success in small- and large-animal studies. This success is significantly impacted by microcapsule morphology and encapsulation efficiency. Since hundreds of thousands of microcapsules are generated during the process, characterization of encapsulated islets without the help of some degree of automation would be difficult, time-consuming, and error prone due to inherent observer bias. We have developed an image analysis algorithm that can analyze hundreds of microencapsulated islets and characterize their size, shape, circularity, and distortion with minimal observer bias. This algorithm can be easily adapted to similar nano- or microencapsulation technologies to implement stricter quality control and improve biomaterial device design and success.
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http://dx.doi.org/10.3727/096368916X693446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657713PMC
May 2017

Prostate magnetic resonance imaging findings in patients treated for testosterone deficiency while on active surveillance for low-risk prostate cancer.

Urol Oncol 2016 12 22;34(12):530.e9-530.e14. Epub 2016 Sep 22.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY. Electronic address:

Objective: To investigate the multiparametric prostate magnetic resonance imaging (mpMRI) findings in patients treated with testosterone replacement therapy (TRT) while on active surveillance for low-risk prostate cancer.

Methods: We retrospectively reviewed 12 patients who underwent mpMRI before and after TRT while on active surveillance. Changes in serum testosterone level, prostate-specific antigen (PSA), prostate biopsy findings, prostate volume, and Prostate Imaging Reporting and Data System Version 2 (PI-RADSv2) score before and after TRT were summarized.

Results: After TRT, there was a significant increase in serum testosterone (516.5ng/dl vs. 203.0ng/dl), PSA (4.2ng/ml vs. 3.3ng/ml), and prostate volume (55.2cm vs. 39.4cm). In total, 2 patients had biopsy progression during the study period. The PI-RADSv2 scores before and after TRT were unchanged in 10/12 patients; none of these demonstrated biopsy progression on post-TRT. The PI-RADSv2 scores increased after TRT in 2/12 patients; both showed Gleason score upgrade on follow-up biopsy. Of these 2 patients, 1 patient underwent radical treatment due to clinical progression. The area under the curve for detecting biopsy progression calculated from PI-RADSv2 score after TRT was 0.90, which was better than that calculated from post-TRT PSA level (0.48).

Conclusions: After TRT, mpMRI findings remained stable in patients without biopsy progression, whereas PI-RADSv2 score increase was identified in patients with Gleason score upgrade on follow-up biopsy.
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http://dx.doi.org/10.1016/j.urolonc.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434455PMC
December 2016

Impact of donor age and weaning status on pancreatic exocrine and endocrine tissue maturation in pigs.

Xenotransplantation 2015 Sep-Oct;22(5):356-67

Department of Surgery, University of California Irvine, Orange, CA, USA.

Background: During the process of islet isolation, pancreatic enzymes are activated and released, adversely affecting islet survival and function. We hypothesize that the exocrine component of pancreases harvested from pre-weaned juvenile pigs is immature and hence pancreatic tissue from these donors is protected from injury during isolation and prolonged tissue culture.

Methods: Biopsy specimens taken from pancreases harvested from neonatal (5-10 days), pre-weaned juvenile (18-22 days), weaned juvenile (45-60 days), and young adult pigs (>90 days) were fixed and stained with hematoxylin and eosin. Sections were examined under a fluorescent microscope to evaluate exocrine zymogen fluorescence intensity (ZFI) and under an electron microscope to evaluate exocrine zymogen granule density (ZGD).

Results: Exocrine content estimation showed significantly lower ZFI and ZGD in juvenile pig pancreases (1.5 ± 0.04 U/μm(2) , ZFI; 1.03 ± 0.07 × 10(3) /100 μm(2) , ZGD) compared to young adult pigs (2.4 ± 0.05U/μm(2) , ZFI; 1.53 ± 0.08 × 10(3) /100 μm(2) ZGD). Islets in juvenile pig pancreases were on average smaller (105.2 ± 11.2 μm) than islets in young adult pigs (192 ± 7.7 μm), but their insulin content was comparable (80.9 ± 2.2% juvenile; 84.2 ± 0.3% young adult, P > 0.05). All data expressed as mean ± SEM.

Conclusion: Porcine islet xenotransplantation continues to make strides toward utilization in clinical trials of type 1 diabetes. Porcine donor age and weaning status influence the extent of exocrine maturation of the pancreas. Juvenile porcine pancreases may represent an alternative donor source for islet xenotransplantation as their exocrine component is relatively immature; this preserves islet viability during extended tissue culture following isolation.
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http://dx.doi.org/10.1111/xen.12184DOI Listing
June 2016

Juvenile porcine islets can restore euglycemia in diabetic athymic nude mice after xenotransplantation.

Transplantation 2015 Apr;99(4):710-6

1 Department of Surgery, University of California Irvine, Orange, CA. 2 Department of Biomedical Engineering, University of California, Irvine, Irvine, CA.

Background: Porcine islet xenotransplantation has been demonstrated in many animal studies to cure experimentally induced diabetes. However, several issues currently impede the translation of porcine islet xenotransplantation to sustained insulin independence clinically. Although adult pigs have mature islets that secrete insulin in response to a glucose challenge, and are physiologically similar to humans, there are logistical considerations with adult porcine tissue that are not present with juvenile porcine tissue. To circumvent these issues, we have identified 18- to 21-day-old preweaned juvenile pigs as islet donors as we have previously demonstrated superior islet yields and function from juvenile pigs using our islet isolation protocols.

Methods: We evaluated the efficacy of islets isolated from 18- to 24-day-old Yorkshire swine in vitro using a standard glucose-stimulated insulin response assay, and in vivo after xenotransplantation under the kidney capsule of streptozotocin-induced 8- to 10-week-old male athymic nude mice. The mice were monitored for a period of 60 days after transplantation, after which the grafts were explanted and analyzed.

Results: Diabetic athymic nude mice transplanted with 1500 to 3000 islet equivalents (IEq) of islets achieved sustained normoglycemia for up to 60 days after islet transplantation. When the grafts were explanted with the kidney, a rapid return to hyperglycemia was observed.

Conclusions: Efficacy and dose-titration studies evaluating these islets in immunocompetent and nonobese diabetic mouse models are underway. The results of these studies will permit application for nonhuman primate and pivotal clinical trials in human diabetic patients in the near future.
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http://dx.doi.org/10.1097/TP.0000000000000667DOI Listing
April 2015

Islet and stem cell encapsulation for clinical transplantation.

Rev Diabet Stud 2014 10;11(1):84-101. Epub 2014 May 10.

Department of Surgery, University of California Irvine, Orange, CA 92868, USA.

Over the last decade, improvements in islet isolation techniques have made islet transplantation an option for a certain subset of patients with long-standing diabetes. Although islet transplants have shown improved graft function, adequate function beyond the second year has not yet been demonstrated, and patients still require immunosuppression to prevent rejection. Since allogeneic islet transplants have experienced some success, the next step is to improve graft function while eliminating the need for systemic immunosuppressive therapy. Biomaterial encapsulation offers a strategy to avoid the need for toxic immunosuppression while increasing the chances of graft function and survival. Encapsulation entails coating cells or tissue in a semipermeable biocompatible material that allows for the passage of nutrients, oxygen, and hormones while blocking immune cells and regulatory substances from recognizing and destroying the cell, thus avoiding the need for systemic immunosuppressive therapy. Despite advances in encapsulation technology, these developments have not yet been meaningfully translated into clinical islet transplantation, for which several factors are to blame, including graft hypoxia, host inflammatory response, fibrosis, improper choice of biomaterial type, lack of standard guidelines, and post-transplantation device failure. Several new approaches, such as the use of porcine islets, stem cells, development of prevascularized implants, islet nanocoating, and multilayer encapsulation, continue to generate intense scientific interest in this rapidly expanding field. This review provides a comprehensive update on islet and stem cell encapsulation as a treatment modality in type 1 diabetes, including a historical outlook as well as current and future research avenues.
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http://dx.doi.org/10.1900/RDS.2014.11.84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295801PMC
May 2015

Encapsulated islet transplantation: strategies and clinical trials.

Immune Netw 2013 Dec 20;13(6):235-9. Epub 2013 Dec 20.

Department of Surgery, University of California Irvine, CA 92868, USA. ; Department of Biomedical Engineering, University of California Irvine, CA 92868, USA.

Encapsulation of tissue has been an area of intense research with a myriad number of therapeutic applications as diverse as cancer, tissue regeneration, and diabetes. In the case of diabetes, transplantation of pancreatic islets of Langerhans containing insulin-producing beta cells has shown promise toward a cure. However, anti-rejection therapy that is needed to sustain the transplanted tissue has numerous adverse effects, and the islets might still be damaged by immune processes. Furthermore, the profound scarcity of healthy human donor organs restricts the availability of islets for transplant. Islet encapsulation allows the protection of this tissue without the use of toxic medications, while also expanding the donor pool to include animal sources. Before the widespread application of this therapy, there are still issues that need to be resolved. There are many materials that can be used, differing shapes and sizes of capsules, and varied sources of islets to name a few variables that need to be considered. In this review, the current options for capsule generation, past animal and human studies, and future directions in this area of research are discussed.
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http://dx.doi.org/10.4110/in.2013.13.6.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875781PMC
December 2013

Noninvasive evaluation of the vascular response to transplantation of alginate encapsulated islets using the dorsal skin-fold model.

Biomaterials 2014 Jan 29;35(3):891-8. Epub 2013 Oct 29.

Department of Surgery, University of California Irvine, Orange, CA 92868, USA.

Alginate encapsulation reduces the risk of transplant rejection by evading immune-mediated cell injury and rejection; however, poor vascular perfusion results in graft failure. Since existing imaging models are incapable of quantifying the vascular response to biomaterial implants after transplantation, in this study, we demonstrate the use of in vivo laser speckle imaging (LSI) and wide-field functional imaging (WiFI) to monitor the microvascular environment surrounding biomaterial implants. The vascular response to two islet-containing biomaterial encapsulation devices, alginate microcapsules and a high-guluronate alginate sheet, was studied and compared after implantation into the mouse dorsal window chamber (N = 4 per implant group). Images obtained over a 14-day period using LSI and WiFI were analyzed using algorithms to quantify blood flow, hemoglobin oxygen saturation and vascular density. Using our method, we were able to monitor the changes in the peri-implant microvasculature noninvasively without the use of fluorescent dyes. Significant changes in blood flow, hemoglobin oxygen saturation and vascular density were noted as early as the first week post-transplant. The dorsal window chamber model enables comparison of host responses to transplanted biomaterials. Future experiments will study the effect of changes in alginate composition on the vascular and immune responses.
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http://dx.doi.org/10.1016/j.biomaterials.2013.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057612PMC
January 2014
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