Publications by authors named "Mohamed I Khalil"

13 Publications

  • Page 1 of 1

Discovery and Function of B-Cell IgD Low (BD) B Cells in Immune Tolerance.

J Mol Biol 2021 Jan 29;433(1):166584. Epub 2020 Jun 29.

Versiti Blood Research Institute, Milwaukee, WI, USA; Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI, USA. Electronic address:

It is now appreciated that in addition to their role in humoral immunity, B cells also exert regulatory mechanisms that lead to attenuation of inflammatory responses. The concept of B-cell regulation became well recognized when mice deficient in B cells due to genetic disruption were shown to be refractory to recovery from the signs of experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. This seminal study spurred the search for B-cell regulatory phenotypes and mechanisms of action. Our approach was to utilize differential B-cell depletion with anti-CD20 to retain B cells whose presence were required to achieve EAE recovery. Utilizing flow cytometry, adoptive cell therapy and genetic approaches, we discovered a new B-cell subset that, upon adoptive transfer into B cell-deficient mice, was sufficient to promote EAE recovery. This B-cell subset is IgM, but due to low/negative IgD cell surface expression, it was named B-cell IgD low (BD). Mechanistically, we found that in the absence of BD, the absolute cell number of CD4Foxp3 T regulatory cells (Treg), essential for immune tolerance, was significantly reduced. Furthermore, we found that BD expression of glucocorticoid-induced tumor necrosis factor ligand (GITRL) was essential for induction of Treg proliferation and maintenance of their homeostasis. Thus, we have identified a new B-cell subset that is critical for immunological tolerance through interactions with Treg.
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http://dx.doi.org/10.1016/j.jmb.2020.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770094PMC
January 2021

Mature IgD B cells maintain tolerance by promoting regulatory T cell homeostasis.

Nat Commun 2019 01 14;10(1):190. Epub 2019 Jan 14.

Blood Research Institute, Versiti Wisconsin, Milwaukee, WI, USA.

A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgD expression maintains tolerance by, at least in part, promoting CD4Foxp3 regulatory T cell homeostatic expansion via glucocorticoid-induced tumor necrosis factor receptor ligand, or GITRL. Cell surface phenotyping, transcriptome analysis and developmental study data show that B cells expressing IgD at a low level (BD) are a novel population of mature B cells that emerge in the spleen from the transitional-2 stage paralleling the differentiation of follicular B cells. The cell surface phenotype and regulatory function of BD are highly suggestive that they are a new B cell subset. Human splenic and peripheral blood IgD B cells also exhibit BD regulatory activity, rendering them of therapeutic interest.
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http://dx.doi.org/10.1038/s41467-018-08122-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331566PMC
January 2019

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication.

Virology 2016 May 23;492:82-91. Epub 2016 Feb 23.

Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA, United States.

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.
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http://dx.doi.org/10.1016/j.virol.2016.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826839PMC
May 2016

Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

Int J Parasitol 2016 Mar 31;46(3):205-212. Epub 2015 Dec 31.

Department of Internal Medicine, Division of Infectious Disease, Stanford University, Stanford, CA 94305, USA; Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA. Electronic address:

Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.
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http://dx.doi.org/10.1016/j.ijpara.2015.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767557PMC
March 2016

Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein.

Virology 2015 Nov 23;485:47-57. Epub 2015 Jul 23.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States.

The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1-4 bound to IE62 and amino acids 238-258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner.
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http://dx.doi.org/10.1016/j.virol.2015.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619144PMC
November 2015

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

Virology 2015 Jul 17;481:179-86. Epub 2015 Mar 17.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States.

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.
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http://dx.doi.org/10.1016/j.virol.2015.02.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437856PMC
July 2015

Epidemiology of neural tube defects.

Saudi Med J 2014 Dec;35 Suppl 1:S29-35

Department of Pediatrics, Security Forces Hospital, Riyadh 11481, Kingdom of Saudi Arabia. E-mail:

Objective: To find the prevalence of neural tube defects (NTDs), and compare the findings with local and international data, and highlight the important role of folic acid supplementation and flour fortification with folic acid in preventing NTDs.

Methods: This is a retrospective study of data retrieved from the medical records of live newborn infants admitted to the Neonatal Intensive Care Unit (NICU), Security Forces Hospital (SFH), Riyadh, Saudi Arabia with NTDs spanning 14 years (1996-2009). All pregnant women on their first antenatal visit to the primary care clinic were prescribed folic acid 0.5 mg daily, or 5 mg if there is a family history of NTD. The pre-fortification prevalence is compared to post-fortification, before and after excluding syndromic, genetic, and chromosomal causes. The results were compared with reports from other parts of Saudi Arabia and internationally, through a literature search using MEDLINE.

Results: The prevalence of NTDs during the period was 1.2 per 1000 live births. The pre-fortification of flour with folic acid prevalence was 1.46 per 1000 live births. The post-fortification prevalence was 1.05 (p=0.103). After excluding syndromic, genetic, and chromosomal causes from calculation of the prevalence, there was a significant reduction in the prevalence, from 1.46 to 0.81 per 1000 live births (p=0.0088). Syndromic, genetic, and chromosomal causes were identified in 20 cases (19.4%). Only 2% of mothers received preconception folic acid, and only 10% of them received it during the first 4 weeks of gestation.

Conclusion: Despite the implementation of fortification of flour with folic acid since 2001, the prevalence of NTDs in the Kingdom of Saudi Arabia is still high. This is due to the impact of genetic, syndromic, and chromosomal causes of NTD not preventable by folic acid. Other factors like unplanned pregnancy and lack of awareness of the role of folic acid in preventing nonsyndromic causes, play a significant role.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362102PMC
December 2014

Cellular transcription factor YY1 mediates the varicella-zoster virus (VZV) IE62 transcriptional activation.

Virology 2014 Jan 12;449:244-53. Epub 2013 Dec 12.

Department of Microbiology and Immunology and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, NY, United States.

Several cellular transcription factors have been shown to be involved in IE62-mediated activation. The YY1 cellular transcription factor has activating and repressive effects on gene transcription. Analysis of the VZV genome revealed 19 postulated YY1 binding sites located within putative promoters of 16 VZV genes. Electrophoretic mobility shift assays (EMSA) confirmed the binding of YY1 to ORF10, ORF28/29 and gI promoters and the mutation of these binding sites inhibited YY1 binding and the promoter activation by IE62 alone or following VZV infection. Mutation of the ORF28/29 YY1 site in the VZV genome displayed insignificant influence on virus growth in melanoma cells; but it inhibited the virus replication significantly at day 5 and 6 post infection in HELF cells. This work suggests a novel role for the cellular factor YY1 in VZV replication through the mediation of IE62 activation of viral gene expression.
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http://dx.doi.org/10.1016/j.virol.2013.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901949PMC
January 2014

Regulation of the varicella-zoster virus ORF3 promoter by cellular and viral factors.

Virology 2013 Jun 21;440(2):171-81. Epub 2013 Mar 21.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, United States.

The varicella zoster virus (VZV) immediate early 62 protein (IE62) activates most if not all identified promoters of VZV genes and also some minimum model promoters that contain only a TATA box element. Analysis of the DNA elements that function in IE62 activation of the VZV ORF3 promoter revealed that the 100 nucleotides before the translation start site of the ORF3 gene contains the promoter elements. This promoter lacks any functional TATA box element. Cellular transcription factors Sp1, Sp3 and YY1 bind to the promoter, and mutation of their binding sites inhibited ORF3 gene expression. VZV regulatory proteins, IE63 and ORF29, ORF61 and ORF10 proteins inhibited IE62-mediated activation of this promoter. Mutation of the Sp1/Sp3 binding site in the VZV genome did not alter VZV replication kinetics. This work suggests that Sp family proteins contribute to the activation of VZV promoters by IE62 in the absence of functional TATA box.
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http://dx.doi.org/10.1016/j.virol.2013.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640663PMC
June 2013

An Sp1/Sp3 site in the downstream region of varicella-zoster virus (VZV) oriS influences origin-dependent DNA replication and flanking gene transcription and is important for VZV replication in vitro and in human skin.

J Virol 2012 Dec 29;86(23):13070-80. Epub 2012 Aug 29.

Department of Microbiology and Immunology, and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, New York, USA.

The distribution and orientation of origin-binding protein (OBP) sites are the main architectural contrasts between varicella-zoster virus (VZV) and herpes simplex virus (HSV) origins of DNA replication (oriS). One important difference is the absence of a downstream OBP site in VZV, raising the possibility that an alternative cis element may replace its function. Our previous work established that Sp1, Sp3, and YY1 bind to specific sites within the downstream region of VZV oriS; we hypothesize that one or both of these sites may be the alternative cis element(s). Here, we show that the mutation of the Sp1/Sp3 site decreases DNA replication and transcription from the adjacent ORF62 and ORF63 promoters following superinfection with VZV. In contrast, in the absence of DNA replication or in transfection experiments with ORF62, only ORF63 transcription is affected. YY1 site mutations had no significant effect on either process. Recombinant viruses containing these mutations were then constructed. The Sp1/Sp3 site mutant exhibited a significant decrease in virus growth in MeWo cells and in human skin xenografts, while the YY1 site mutant virus grew as well as the wild type in MeWo cells, even showing a late increase in VZV replication in skin xenografts following infection. These results suggest that the Sp1/Sp3 site plays an important role in both VZV origin-dependent DNA replication and ORF62 and ORF63 transcription and that, in contrast to HSV, these events are linked during virus replication.
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http://dx.doi.org/10.1128/JVI.01538-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497629PMC
December 2012

A sequence within the varicella-zoster virus (VZV) OriS is a negative regulator of DNA replication and is bound by a protein complex containing the VZV ORF29 protein.

J Virol 2011 Dec 21;85(23):12188-200. Epub 2011 Sep 21.

Department of Microbiology and Immunology and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, New York 14214, USA.

The architecture of the varicella-zoster virus (VZV) origin of DNA replication (OriS) differs significantly from that of the herpes simplex virus (HSV) DNA replication origin. Novel aspects of the VZV OriS include a GA-rich region, three binding sites for the VZV origin-binding protein (OBP) all on the same strand and oriented in the same direction, and a partial OBP binding site of unknown function. We have designated this partial binding site Box D and have investigated the role it plays in DNA replication and flanking gene expression. This has been done with a model system using a replication-competent plasmid containing OriS and a replication- and transcription-competent dual-luciferase reporter plasmid containing both the OriS and the intergenic region between VZV open reading frames (ORFs) 62 and 63. We have found that (i) Box D is a negative regulator of DNA replication independent of flanking gene expression, (ii) the mutation of Box D results in a decrease in flanking gene expression, thus a sequence within the VZV OriS affects transcription, which is in contrast to results reported for HSV-1, (iii) there is a specific Box D complex formed with infected cell extracts in electrophoretic mobility shift assay experiments, (iv) supershift assays show that this complex contains the VZV ORF29 single-strand DNA-binding protein, and (v) the formation of this complex is dependent on the presence of CGC motifs in Box D and its downstream flanking region. These findings show that the VZV ORF29 protein, while required for DNA replication, also plays a novel role in the suppression of that process.
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http://dx.doi.org/10.1128/JVI.05501-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209344PMC
December 2011

A method to improve the effectiveness of the Bakri balloon for management of postpartum hemorrhage at cesarean.

Int J Gynaecol Obstet 2011 Nov 14;115(2):198-200. Epub 2011 Sep 14.

Department of Obstetrics and Gynecology, Security Forces Hospital, Riyadh, Saudi Arabia.

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http://dx.doi.org/10.1016/j.ijgo.2011.04.018DOI Listing
November 2011

Cellular transcription factors Sp1 and Sp3 suppress varicella-zoster virus origin-dependent DNA replication.

J Virol 2008 Dec 24;82(23):11723-33. Epub 2008 Sep 24.

Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, The State University of New York, Buffalo, New York 14214, USA.

The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.
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http://dx.doi.org/10.1128/JVI.01322-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583665PMC
December 2008