Publications by authors named "Therwa Hamza"

15 Publications

  • Page 1 of 1

Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling.

BMC Immunol 2021 Mar 25;22(1):23. Epub 2021 Mar 25.

Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, 650 W Baltimore Street, Baltimore, MD, 21201, USA.

Background: Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria's outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro.

Results: Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL.

Conclusion: TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.
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http://dx.doi.org/10.1186/s12865-021-00409-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995782PMC
March 2021

A probiotic yeast-based immunotherapy against infection.

Sci Transl Med 2020 10;12(567)

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA.

Antibiotic-resistant is an anaerobic Gram-positive bacterium that colonizes the colon and is responsible for more than 29,000 deaths in the United States each year. Hence, infection (CDI) poses an urgent threat to public health. Antibody-mediated neutralization of TcdA and TcdB toxins, the major virulence factors of CDI, represents an effective strategy to combat the disease without invoking antibiotic resistance. However, current antitoxin approaches are mostly based on parenteral infusion of monoclonal antibodies that are costly, narrow spectrum, and not optimized against the intestinal disease. Here, we engineered probiotic to constitutively secrete a single tetra-specific antibody that potently and broadly neutralized both toxins and demonstrated protection against primary and recurrent CDI in both prophylactic and therapeutic mouse models of disease. This yeast immunotherapy is orally administered, can be used concurrently with antibiotics, and may have potential as a prophylactic against CDI risk and as a therapeutic for patients with CDI.
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http://dx.doi.org/10.1126/scitranslmed.aax4905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692727PMC
October 2020

Structure of the full-length Clostridium difficile toxin B.

Nat Struct Mol Biol 2019 08 15;26(8):712-719. Epub 2019 Jul 15.

Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.
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http://dx.doi.org/10.1038/s41594-019-0268-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684407PMC
August 2019

Host-targeted niclosamide inhibits C. difficile virulence and prevents disease in mice without disrupting the gut microbiota.

Nat Commun 2018 12 7;9(1):5233. Epub 2018 Dec 7.

Molecular Medicine, Hospital for Sick Children, 686 Bay St., Toronto, ON, M5G 0A4, Canada.

Clostridium difficile is the leading cause of nosocomial diarrhea and colitis in the industrialized world. Disruption of the protective gut microbiota by antibiotics enables colonization by multidrug-resistant C. difficile, which secrete up to three different protein toxins that are responsible for the gastrointestinal sequelae. Oral agents that inhibit the damage induced by toxins, without altering the gut microbiota, are urgently needed to prevent primary disease and break the cycle of antibiotic-induced disease recurrence. Here, we show that the anthelmintic drug, niclosamide, inhibits the pathogenesis of all three toxins by targeting a host process required for entry into colonocytes by each toxin. In mice infected with an epidemic strain of C. difficile, expressing all three toxins, niclosamide reduced both primary disease and recurrence, without disrupting the diversity or composition of the gut microbiota. Given its excellent safety profile, niclosamide may address an important unmet need in preventing C. difficile primary and recurrent diseases.
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http://dx.doi.org/10.1038/s41467-018-07705-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286312PMC
December 2018

Mice with Inflammatory Bowel Disease are Susceptible to Clostridium difficile Infection With Severe Disease Outcomes.

Inflamm Bowel Dis 2018 02;24(3):573-582

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, Maryland, USA.

Background: Over the past several decades, there has been a significant increase in the incidence of Clostridium difficile infection (CDI) in patients suffering from inflammatory bowel disease (IBD). However, a wild-type animal model is not available to study these comorbid diseases.

Methods: We evaluated the susceptibility to CDI of mice with dextran sulfate sodium salt (DSS)-induced colitis (IBD mice) with or without antibiotic exposure; we examined the histopathology and cytokine response in the concomitant diseases after the model was created.

Results: No CDI occurs in healthy control mice, wherease the incidence of CDI in IBD mice is 40%; however, in IBD mice that received antibiotics, the incidence of CDI is 100% and the disease is accompanied by high levels of toxins in the mouse feces and sera. Compared to IBD and CDI alone, those IBD mice infected with C. difficile have more severe symptoms, toxemia, histopathological damage, and higher mortality. Moreover, several proinflammatory cytokines and chemokines are significantly elevated in the colon tissues from IBD mice infected with C. difficile.

Conclusions: We, for the first time, demonstrate in an animal model that mice with dextran sulfate sodium induced-inflammatory bowel disease are significantly more susceptible to C. difficile infection, and that the bacterial infection led to more severe disease and death. These findings are consistent with clinical observations, thus, the animal model will permit us to study the pathogenesis of these concurrent diseases and to develop therapeutic strategies against the comorbidity of IBD and CDI.
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http://dx.doi.org/10.1093/ibd/izx059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936643PMC
February 2018

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Anaerobe 2017 Dec 12;48:249-256. Epub 2017 Oct 12.

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD, USA. Electronic address:

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.
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http://dx.doi.org/10.1016/j.anaerobe.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792825PMC
December 2017

Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Infection.

J Vaccines Vaccin 2016;7(3). Epub 2016 May 27.

Department of Medicine, Division of Infectious Disease, University of Maryland School of Medicine, Baltimore, Maryland, USA.

is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by -associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in . We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low anti-pilin antibody titers and no protection upon challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of infection, are needed to determine if an anti-T4P vaccine would be protective against infection.
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http://dx.doi.org/10.4172/2157-7560.1000321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927082PMC
May 2016

A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

Biofouling 2016 07;32(6):627-34

a Department of Orthopaedics , West Virginia University , Morgantown , WV , USA.

Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.
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http://dx.doi.org/10.1080/08927014.2016.1172572DOI Listing
July 2016

Nanoparticles targeting to osteoblasts for potential intracellular pathogen elimination.

J Control Release 2015 Sep 19;213:e10-1. Epub 2015 Aug 19.

Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown 26506, USA.

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http://dx.doi.org/10.1016/j.jconrel.2015.05.013DOI Listing
September 2015

Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis.

Pathog Dis 2016 Feb 26;74(1):ftv098. Epub 2015 Oct 26.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA

The Clostridium difficile toxin B is one of the main virulence factors and plays an important role in the pathogenesis of C. difficile infection (CDI). We recently revealed crucial residues in the translocation domain of TcdB for the pore formation and toxin translocation. In this study, we investigated the effects of mutating a critical site involved in pore formation, Leu-1106, to residues that differ in size and polarity (Phe, Ala, Cys, Asp). We observed a broad range of effects on TcdB function in vitro consistent with the role of this site in pore formation and translocation. We show that mice challenged systemically with a lethal dose (LD100) of the most defective mutant (L1106K) showed no symptoms of disease highlighting the importance of this residue and the translocation domain in disease pathogenesis. These findings offer insights into the structure function of the toxin translocation pore, and inform novel therapeutic strategies against CDI.
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http://dx.doi.org/10.1093/femspd/ftv098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882082PMC
February 2016

Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides.

Biochem Biophys Res Commun 2015 Apr 26;459(2):259-263. Epub 2015 Feb 26.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, MD 21201, USA. Electronic address:

Clostridium difficile toxin A and B (TcdA and TcdB) are the major virulence factors of the bacterium, both of which consist of two enzymatic domains: an effector glucosyltransferase domain (GTD) and a cysteine protease domain (CPD) responsible for autocleavage and release of GTD. Although the CPDs from both toxins share a similar structure and mechanism of hexakisphosphate (InsP6)-induced activation, TcdA is substantially less sensitive to the autocleavage as compared with TcdB. In this study, we provided evidence of inter-domain regulation of CPD activity of TcdA and its autoprocessing. The C-terminus combined repetitive oligo peptides (CROPs) of TcdA reduced the accessibility of TcdB CPD to its substrate in a chimeric toxin TxB-Ar, consequently blocking autoprocessing. Moreover, interference of antibodies with the CROPs of full-length TcdA efficiently enhanced its GTD release. In conclusion, by utilizing chimeric toxins and specific antibodies, we identified that the CROPs of TcdA plays a crucial role in controlling the InsP6-mediated activation of CPD and autocleavage of GTD. Our data provides insights on the molecular mode of action of the C. difficile toxins.
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http://dx.doi.org/10.1016/j.bbrc.2015.02.095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426850PMC
April 2015

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Infect Immun 2015 Feb 8;83(2):822-31. Epub 2014 Dec 8.

Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USA

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.
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http://dx.doi.org/10.1128/IAI.02897-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294251PMC
February 2015

Differential responses of osteoblasts and macrophages upon Staphylococcus aureus infection.

BMC Microbiol 2014 Jul 25;14:207. Epub 2014 Jul 25.

Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown 26506, WV, USA.

Background: Staphylococcus aureus (S. aureus) is one of the primary causes of bone infections which are often chronic and difficult to eradicate. Bacteria like S. aureus may survive upon internalization in cells and may be responsible for chronic and recurrent infections. In this study, we compared the responses of a phagocytic cell (i.e. macrophage) to a non-phagocytic cell (i.e. osteoblast) upon S. aureus internalization.

Results: We found that upon internalization, S. aureus could survive for up to 5 and 7 days within macrophages and osteoblasts, respectively. Significantly more S. aureus was internalized in macrophages compared to osteoblasts and a significantly higher (100 fold) level of live intracellular S. aureus was detected in macrophages compared to osteoblasts. However, the percentage of S. aureus survival after infection was significantly lower in macrophages compared to osteoblasts at post-infection days 1-6. Interestingly, macrophages had relatively lower viability in shorter infection time periods (i.e. 0.5-4 h; significant at 2 h) but higher viability in longer infection time periods (i.e. 6-8 h; significant at 8 h) compared to osteoblasts. In addition, S. aureus infection led to significant changes in reactive oxygen species production in both macrophages and osteoblasts. Moreover, infected osteoblasts had significantly lower alkaline phosphatase activity at post-infection day 7 and infected macrophages had higher phagocytosis activity compared to non-infected cells.

Conclusions: S. aureus was found to internalize and survive within osteoblasts and macrophages and led to differential responses between osteoblasts and macrophages. These findings may assist in evaluation of the pathogenesis of chronic and recurrent infections which may be related to the intracellular persistence of bacteria within host cells.
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http://dx.doi.org/10.1186/s12866-014-0207-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116603PMC
July 2014

Unique antimicrobial effects of platelet-rich plasma and its efficacy as a prophylaxis to prevent implant-associated spinal infection.

Adv Healthc Mater 2013 Sep 27;2(9):1277-84. Epub 2013 Feb 27.

Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown, WV 26506, USA; Department of Orthopaedics, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.

Platelet-rich-plasma (PRP) has attracted great attention and has been increasingly used for a variety of clinical applications including orthopedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, and sports medicine. However, very little is known about the antimicrobial activities of PRP. PRP is found to have antimicrobial properties both in vitro and in vivo. In vitro, the antimicrobial properties of PRP are bacterial-strain-specific and time-specific: PRP significantly (80-100 fold reduction in colony-forming units) inhibits the growth of methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A streptococcus, and Neisseria gonorrhoeae within the first few hours but it has no significant antimicrobial properties against E. coli and Pseudomonas. The antimicrobial properties of PRP also depend on the concentration of thrombin. In vivo, an implant-associated spinal infection rabbit model is established and used to evaluate the antimicrobial and wound-healing properties of PRP. Compared to the infection controls, PRP treatment results in significant reduction in bacterial colonies in bone samples at all time points studied (i.e. 1, 2, and 3 weeks) and significant increase in mineralized tissues (thereby better bone healing) at postoperative weeks 2 and 3. PRP therefore may be a useful adjunct strategy against postoperative implant-associated infections.
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http://dx.doi.org/10.1002/adhm.201200465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774283PMC
September 2013

Interleukin 12 a key immunoregulatory cytokine in infection applications.

Int J Mol Sci 2010 Feb 26;11(3):789-806. Epub 2010 Feb 26.

Department of Orthopaedics, West Virginia University, Morgantown, 26506, USA.

Interleukin 12 (termed IL-12p70 and commonly designated IL-12) is an important immunoregulatory cytokine that is produced mainly by antigen-presenting cells. The expression of IL-12 during infection regulates innate responses and determines the type of adaptive immune responses. IL-12 induces interferon-gamma (IFN-gamma) production and triggers CD4(+) T cells to differentiate into type 1 T helper (Th1) cells. Studies have suggested that IL-12 could play a vital role in treating many diseases, such as viral and bacterial infections and cancers. The unique heterodimeric structure, which IL-12 shares with its family members including IL-23, IL-27, and IL-35, has recently brought more attention to understanding the mechanisms that regulate the functions of IL-12. This article describes the structure and biological activities of IL-12 in both the innate and adaptive arms of the immune system, and discusses the applications of IL-12 in treating and preventing infections.
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http://dx.doi.org/10.3390/ijms11030789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869233PMC
February 2010