Establishment Of Chronic Infection Research Articles

Antigenic cooperation in viral populations: Transformation of functions of intra-host viral variants

In this paper we study intra-host viral adaptation by antigenic cooperation - a mechanism of immune escape that serves as an alternative to the standard mechanism of escape by continuous genomic diversification and allows to explain a number of experimental observations associated with the establishment of chronic infections by highly mutable viruses. Within this mechanism, the topology of a cross-immunoreactivity network forces intra-host viral variants to specialize for complementary roles and adapt to host’s immune response as a quasi-social ecosystem. Here we study dynamical changes in immune adaptation caused by evolutionary and epidemiological events. First, we show that the emergence of a viral variant with altered antigenic features may result in a rapid re-arrangement of the viral ecosystem and a change in the roles played by existing viral variants. In particular, it may push the population under immune escape by genomic diversification towards the stable state of adaptation by antigenic cooperation. Next, we study the effect of a viral transmission between two chronically infected hosts, which results in merging of two intra-host viral populations in the state of stable immune-adapted equilibrium. In this case, we also describe how the newly formed viral population adapts to the host’s environment by changing the functions of its members. The results are obtained analytically for minimal cross-immunoreactivity networks and numerically for larger populations.

Protective immunity induced by DNA vaccine containing TgGRA35, TgGRA42, and TgGRA43 against Toxoplasma gondii infection in Kunming mice.

Toxoplasma gondii can cause congenital infection and abortion in humans and warm-blooded animals. T. gondii dense granule proteins, GRA35, GRA42, and GRA43, play a critical role in the establishment of chronic infection. However, their potential to induce protective immunity against T. gondii infection remains unexplored. This study aimed to test the efficacy of a DNA vaccine encompassing GRA35, GRA42, and GRA43 in inducing protective immunity against the highly virulent T. gondii RH strain (type I) and the brain cyst-forming PRU strain (type II). The eukaryotic plasmids pVAX-GRA35, pVAX-GRA42, and pVAX-GRA43 were constructed and formulated into two- or three-gene cocktail DNA vaccines. The indirect immunofluorescence assay (IFA) was used to analyze their expression and immunogenicity. Mice were immunized with a single-gene, two-genes, or multicomponent eukaryotic plasmid, intramuscularly. We assessed antibody levels, cytotoxic T-cell (CTL) responses, cytokines, and lymphocyte surface markers by using flow cytometry. Additionally, mouse survival and cyst numbers in the brain of mice challenged 1 to 2 months postvaccination were determined. Specific humoral and cellular immune responses were elicited in mice immunized with single-, two-, or three-gene cocktail DNA vaccine, as indicated by significant increases in serum antibody concentrations of total IgG, IgG2a/IgG1 ratio, cytokine levels (IFN-γ, IL-2, IL-12, IL-4, and IL-10), lymphocyte proliferation, lymphocyte populations (CD4+ and CD8+ T lymphocytes), CTL activities, and survival, as well as decreased brain cysts, in comparison with control mice. Moreover, compared with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43, multicomponent DNA vaccine with three genes (pVAX-GRA35 + pVAX-GRA42 + pVAX-GRA43) induced the higher humoral and cellular immune responses, including serum antibody concentrations, cytokine levels, lymphocyte proliferation, lymphocyte populations, CTL activities and survival, resulting in prolonged survival time and reduced brain cyst loads. Furthermore, mice immunized with pVAX-GRA35 + pVAX-GRA42, pVAX-GRA42 + pVAX-GRA43, or pVAX-GRA35 + pVAX-GRA43 showed greater Th1 immune responses and protective efficacy than the single-gene-vaccinated groups. These results demonstrate that TgGRA35, TgGRA42, or TgGRA43 are vaccine candidates against T. gondii infection, and the three-gene DNA vaccine cocktail conferred the strongest protection against T. gondii infection.

Within-Host Evolution of Bacterial Pathogens in Acute and Chronic Infection.

Bacterial pathogens undergo remarkable adaptive change in response to the selective forces they encounter during host colonization and infection. Studies performed over the past few decades have demonstrated that many general evolutionary processes can be discerned during the course of host adaptation, including genetic diversification of lineages, clonal succession events, convergent evolution, and balanced fitness trade-offs. In some cases, elevated mutation rates resulting from mismatch repair or proofreading deficiencies accelerate evolution, and active mobile genetic elements or phages may facilitate genome plasticity. The host immune response provides another critical component of the fitness landscapes guiding adaptation, and selection operating on pathogens at this level may lead to immune evasion and the establishment of chronic infection. This review summarizes recent advances in this field, with a special focus on different forms of bacterial genome plasticity in the context of infection, and considers clinical consequences of adaptive changes for the host.

Biologically Relevant Murine Models of Chronic Pseudomonas aeruginosa Respiratory Infection.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen and the leading cause of infection in patients with cystic fibrosis (CF). The ability of P. aeruginosa to evade host responses and develop into chronic infection causes significant morbidity and mortality. Several mouse models have been developed to study chronic respiratory infections induced by P. aeruginosa, with the bead agar model being the most widely used. However, this model has several limitations, including the requirement for surgical procedures and high mortality rates. Herein, we describe novel and adapted biologically relevant models of chronic lung infection caused by P. aeruginosa. Three methods are described: a clinical isolate infection model, utilising isolates obtained from patients with CF; an incomplete antibiotic clearance model, leading to bacterial bounce-back; and the establishment of chronic infection; and an adapted water bottle chronic infection model. These models circumvent the requirement for a surgical procedure and, importantly, can be induced with clinical isolates of P. aeruginosa and in wild-type mice. We also demonstrate successful induction of chronic infection in the transgenic βENaC murine model of CF. We envisage that the models described will facilitate the investigations of host and microbial factors, and the efficacy of novel antimicrobials, during chronic P. aeruginosa respiratory infections.

Deciphering the roles of myeloid derived suppressor cells in viral oncogenesis.

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of myeloid cells derived from monocyte and granulocyte precursors. They are pathologically expanded in conditions of ongoing inflammation where they function to suppress both innate and adaptive immunity. They are subdivided into three distinct subsets: monocytic (M-) MDSC, polymorphonuclear (or neutrophilic) (PMN-) MDSC and early-stage (e-) MDSC that may exhibit differential function in different pathological scenarios. However, in cancer they are associated with inhibition of the anti-tumour immune response and are universally associated with a poor prognosis. Seven human viruses classified as Group I carcinogenic agents are jointly responsible for nearly one fifth of all human cancers. These viruses represent a large diversity of species, including DNA, RNA and retroviridae. They include the human gammaherpesviruses (Epstein Barr virus (EBV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV), members of the high-risk human papillomaviruses (HPVs), hepatitis B and C (HBV, HCV), Human T cell leukaemia virus (HTLV-1) and Merkel cell polyomavirus (MCPyV). Each of these viruses encode an array of different oncogenes that perturb numerous cellular pathways that ultimately, over time, lead to cancer. A prerequisite for oncogenesis is therefore establishment of chronic infection whereby the virus persists in the host cells without being eradicated by the antiviral immune response. Although some of the viruses can directly modulate the immune response to enable persistence, a growing body of evidence suggests the immune microenvironment is modulated by expansions of MDSCs, driven by viral persistence and oncogenesis. It is likely these MDSCs play a role in loss of immune recognition and function and it is therefore essential to understand their phenotype and function, particularly given the increasing importance of immunotherapy in the modern arsenal of anti-cancer therapies. This review will discuss the role of MDSCs in viral oncogenesis. In particular we will focus upon the mechanisms thought to drive the MDSC expansions, the subsets expanded and their impact upon the immune microenvironment. Importantly we will explore how MDSCs may modulate current immunotherapies and their impact upon the success of future immune-based therapies.

T cell-extrinsic IL-1 signaling controls long-term gammaherpesvirus infection by suppressing viral reactivation

Gammaherpesviruses establish life-long infection in over 95% of adults and are associated with several cancers, including B cell lymphomas. Using the murine gammaherpesvirus 68 (MHV68) animal model, we previously showed a pro-viral role of Interleukin-1 (IL-1) signaling that supported viral reactivation during the establishment of chronic infection. Unexpectedly, in this study we found that the proviral effects of IL-1 signaling originally observed during the establishment of chronic gammaherpesvirus infection convert to antiviral effects during the long-term stage of infection. Specifically, IL-1 signaling promoted expansion of antiviral CD8+ T cells and control of viral reactivation in the peritoneal cavity of a long-term infected host. Using a novel mouse model of T cell-specific IL-1 signaling deficiency, we found that the antiviral effects of IL-1 signaling were T cell extrinsic. Our study highlights a dynamic nature of host factors that shape the parameters of chronic gammaherpesvirus infection.

Bacterial Culture Underestimates Lung Pathogen Detection and Infection Status in Cystic Fibrosis.

ABSTRACTMicrobiological surveillance of airway secretions is central to clinical care in cystic fibrosis (CF). However, the efficacy of microbiological culture, the diagnostic gold standard for pathogen detection, has been increasingly questioned. Here we compared culture with targeted quantitative PCR (QPCR) for longitudinal detection of 2 key pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Prospectively collected respiratory samples taken from 20 pediatric and 20 adult CF patients over a period of 3-years were analyzed. Patients were eligible if considered free of chronic Pseudomonas infection within 12-months prior to start of study. QPCR revealed high levels of infection with both pathogens not apparent from culture alone. Pseudomonas and Staphylococcus were detected by culture on at least one sampling occasion in 12 and 29 of the patients, respectively. Conversely, both pathogens were detected in all 40 patients by QPCR. Classification of infection status also significantly altered in both pediatric and adult patients, where the number of patients deemed chronically infected with Pseudomonas and Staphylococcus increased from 1 to 28 and 9 to 34, respectively. Overall, Pseudomonas and Staphylococcus infection status classification changed respectively for 36 and 27 of all patients. In no cases did molecular identification lead to a patient being in a less clinically serious infection category. Pathogen detection and infection status classification significantly increased when assessed by QPCR in comparison to culture. This could have implications for clinical care of CF patients, including accuracy of infection diagnosis, relevant and timely antibiotic selection, antimicrobial resistance development, establishment of chronic infection, and cross-infection control.IMPORTANCE Chronic lung infection is the leading cause of morbidity and early mortality for people with cystic fibrosis (pwCF). Microbiological surveillance to detect lung pathogens is recommended as best practise in CF patient care. Here we studied pathogen detection in 40 pwCF over several years. We found that microbiological culture, the diagnostic gold standard, was significantly disparate to targeted culture-independent approaches for detection and determination of chronic infection status of two important pathogens in CF. Pathogen detection was significantly lower by culture and consequently infection status was also misclassified in most cases. In particular, the extent of chronic infection by both P. aeruginosa and S. aureus not realized with culture was striking. Our findings have implications for the development of infection and clinical care of pwCF. Future longitudinal studies with greater patient numbers will be needed to establish the full extent of the clinical implications indicated from this study.

A recombinant protein vaccine encoding Toxoplasma gondii Cyst wall 2 (dense granule protein 47) provides partial protection against acute and chronic T. gondii infection in BALB/c mice

Toxoplasma gondii poses a major threat to economies and public health, and there are still no available vaccines for human against T. gondii infection. T. gondii cyst wall 2 (TgCST2, also known as dense granule protein-47) is a critical molecule in the establishment of chronic infection, making it a potential vaccine candidate. In this research, the recombinant TgCST2 (rTgCST2) was employed to evaluate the protective efficacy of TgCST2 antigen using BALB/c mice model against T. gondii infections via active immunization trials. First, the strong immunogenicity of TgCST2 was indicated by immunoblotting and immunofluorescence, which mean that TgCST2 might elicit robust immune responses in the organism. Then, after triply subcutaneous immunization with rTgCST2/ISA 201 emulsion, high levels of Toxoplasma-specific IgG, IgG1, IgG2a and cytokines (Interferon γ and interleukin 10) further suggested that TgCST2 was a promising immunogenic antigen. More importantly, this antigen could prolong survival in RH strain infected mice and resulted in the lower brain cysts size and number of PRU strain infected mice. These preliminary results demonstrated the immunoprophylactic effects of TgCST2 antigen and will inform new studies in developing subunit recombinant vaccines against T. gondii.

Mucosal Immunity in Cystic Fibrosis.

The highly complex and variable genotype-phenotype relationships observed in cystic fibrosis (CF) have been an area of growing interest since the discovery of the CF transmembrane conductance regulator (CFTR) gene >30 y ago. The consistently observed excessive, yet ineffective, activation of both the innate and adaptive host immune systems and the establishment of chronic infections within the lung, leading to destruction and functional decline, remain the primary causes of morbidity and mortality in CF. The fact that both inflammation and pathogenic bacteria persist despite the introduction of modulator therapies targeting the defective protein, CFTR, highlights that we still have much to discover regarding mucosal immunity determinants in CF. Gene modifier studies have overwhelmingly implicated immune genes in the pulmonary phenotype of the disease. In this context, we aim to review recent advances in our understanding of the innate and adaptive immune systems in CF lung disease.

Bacterial evolution during human infection: Adapt and live or adapt and die.

Microbes are constantly evolving. Laboratory studies of bacterial evolution increase our understanding of evolutionary dynamics, identify adaptive changes, and answer important questions that impact human health. During bacterial infections in humans, however, the evolutionary parameters acting on infecting populations are likely to be much more complex than those that can be tested in the laboratory. Nonetheless, human infections can be thought of as naturally occurring in vivo bacterial evolution experiments, which can teach us about antibiotic resistance, pathogenesis, and transmission. Here, we review recent advances in the study of within-host bacterial evolution during human infection and discuss practical considerations for conducting such studies. We focus on 2 possible outcomes for de novo adaptive mutations, which we have termed “adapt-and-live” and “adapt-and-die.” In the adapt-and-live scenario, a mutation is long lived, enabling its transmission on to other individuals, or the establishment of chronic infection. In the adapt-and-die scenario, a mutation is rapidly extinguished, either because it carries a substantial fitness cost, it arises within tissues that block transmission to new hosts, it is outcompeted by more fit clones, or the infection resolves. Adapt-and-die mutations can provide rich information about selection pressures in vivo, yet they can easily elude detection because they are short lived, may be more difficult to sample, or could be maladaptive in the long term. Understanding how bacteria adapt under each of these scenarios can reveal new insights about the basic biology of pathogenic microbes and could aid in the design of new translational approaches to combat bacterial infections.

Non-Antimicrobial Adjuvant Strategies to Tackle Biofilm-Related Staphylococcus aureus Prosthetic Joint Infections.

Staphylococcus aureus frequently causes community- and hospital-acquired infections. S. aureus attachment followed by biofilm formation on tissues and medical devices plays a significant role in the establishment of chronic infections. Staphylococcal biofilms encase bacteria in a matrix and protect the cells from antimicrobials and the immune system, resulting in infections that are highly resistant to treatment. The biology of biofilms is complex and varies between organisms. In this review, we focus our discussion on S. aureus biofilms and describe the stages of their formation. We particularly emphasize genetic and biochemical processes that may be vulnerable to novel treatment approaches. Against this background, we discuss treatment strategies that have been successful in animal models of S. aureus biofilm-related infection and consider their possible use for the prevention and eradication of biofilm-related S. aureus prosthetic joint infection.

Compensatory evolution of Pseudomonas aeruginosa\u2019s slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.

Immunological Responses to Seoul Orthohantavirus in Experimentally and Naturally Infected Brown Rats (Rattus norvegicus).

To clarify the mechanism of Seoul orthohantavirus (SEOV) persistence, we compared the humoral and cell-mediated immune responses to SEOV in experimentally and naturally infected brown rats. Rats that were experimentally infected by the intraperitoneal route showed transient immunoglobulin M (IgM) production, followed by an increased anti-SEOV immunoglobulin G (IgG) antibody response and maturation of IgG avidity. The level of SEOV-specific cytotoxic T lymphocytes (CTLs) peaked at 6 days after inoculation and the viral genome disappeared from serum. In contrast, naturally infected brown rats simultaneously had a high rate of SEOV-specific IgM and IgG antibodies (28/43). Most of the IgM-positive rats (24/27) had the SEOV genome in their lungs, suggesting that chronic SEOV infection was established in those rats. In female rats with IgG avidity maturation, the viral load in the lungs was decreased. On the other hand, there was no relationship between IgG avidity and viral load in the lungs in male rats. A CTL response was not detected in naturally infected rats. The difference between immune responses in the experimentally and naturally infected rats is associated with the establishment of chronic infection in natural hosts.

Non-neutralizing antibodies protect against chronic LCMV infection by promoting infection of inflammatory monocytes in mice.

Antibodies play an important role in host defense against microorganisms. Besides direct microbicidal activities, antibodies can also provide indirect protection via crosstalk to constituents of the adaptive immune system. Similar to many human chronic viral infections, persistence of Lymphocytic choriomeningitis virus (LCMV) is associated with compromised T‐ and B‐cell responses. The administration of virus‐specific non‐neutralizing antibodies (nnAbs) prior to LCMV infection protects against the establishment of chronic infection. Here, we show that LCMV‐specific nnAbs bind preferentially Ly6Chi inflammatory monocytes (IMs), promote their infection in an Fc‐receptor independent way, and support acquisition of APC properties. By constituting additional T‐cell priming opportunities, IMs promote early activation of virus‐specific CD8 T cells, eventually tipping the balance between T‐cell exhaustion and effector cell differentiation, preventing establishment of viral persistence without causing lethal immunopathology. These results document a beneficial role of IMs in avoiding T‐cell exhaustion and an Fc‐receptor independent protective mechanism provided by LCMV‐specific nnAbs against the establishment of chronic infection.

Anti-biofilm activity of plant derived extracts against infectious pathogen-Pseudomonas aeruginosa PAO1

BackgroundBiofilm forming ability of Pseudomonas aeruginosa make them vulnerable, because it makes them recalcitrant against various antibiotics. Quorum sensing (QS) is cell density based signaling that helps in bacterial cell–cell communication, which regulated various virulence factors such as pigment and biofilm formation that contribute in the establishment of chronic infections. The interruption of QS is one of the effective approach to control various virulence factors. Present study was intended with the aim to authenticate antibiofilm potential in different solvents based extracts of selected medicinal plant species viz. Berginia ciliata, Clematis grata and Clematis viticella traditionally used by the inhabitants of Himalayan region of Pakistan to treat various pathogenic diseases. P. aeruginosa PAO1, an opportunistic pathogen and involves in various life-threatening infections specifically in immune deficient patients was used as a model pathogen. MethodsPlants were extracted in various organic (ethanol, methanol, acetone, ethyl acetate, hexane, chloroform) as well as in aqueous solvents and their ability to inhibit biofilm was measured. Biofilm of PAO1 was grown in Jensen's medium while growing at 30°C and crystal violet assay was performed to assess the biofilm inhibiting activity of plant extracts. ResultsSolvents play a vital role in extraction of plant components and it was found that the plants in various solvents exhibit different activity against the PAO1 biofilm. Comparatively, 1% methanolic extract of B. ciliata (rhizome with skin), showed more than 80% inhibition of biofilm formation without effecting on the growth of the bacterium. Significant correlation between flavonoids content and antibiofilm activity in methanolic extract revealed the contribution of secondary metabolites in P. aeruginosa (PAO1) biofilm inhibition. ConclusionOur study revealed that plants under investigation more specifically B. ciliata could be a potential candidate for drug discovery to treat P. aeruginosa PAO1, induced infectious diseases especially for its biofilm treatment.

Pseudomonas aeruginosa Strain-sharing in Early Infection Among Children With Cystic Fibrosis.

We previously identified Pseudomonas aeruginosa isolates with characteristics typical of chronic infection in some early infections in children with cystic fibrosis (CF), suggesting that these isolates may have been acquired from other patients. Our objective was to define the extent of P. aeruginosa strain-sharing in early CF infections and its impact on antibiotic eradication treatment failure rates. We performed whole genome sequencing on isolates from early pediatric CF pulmonary infections and from the following comparator groups in the same hospital: chronic CF infection, sink drains, sterile site infections, and asymptomatic carriage. Univariate logistic regression was used to assess factors associated with treatment failure. In this retrospective, observational study, 1029 isolates were sequenced. The CF clones strain B and clone C were present. In 70 CF patients with early infections, 14 shared strains infected 29 (41%) patients over 5 years; 16% (n = 14) of infections had mixed strains. In the 70 children, approximately one-third of shared-strain infections were likely due to patient-to-patient transmission. Mixed-strain infections were associated with strain-sharing (odds ratio, 8.50; 95% confidence interval, 2.2-33.4; P = .002). Strain-sharing was not associated with antibiotic eradication treatment failure; however, nosocomial strain transmission was associated with establishment of chronic infection in a CF sibling pair. Although early P. aeruginosa CF infection is thought to reflect acquisition of diverse strains from community reservoirs, we identified frequent early CF strain-sharing that was associated with the presence of mixed strains and instances of possible patient-to-patient transmission.

B Cell-Intrinsic Expression of Interferon Regulatory Factor 1 Supports Chronic Murine Gammaherpesvirus 68 Infection.

Gammaherpesviruses are ubiquitous pathogens that are associated with cancers, including B cell lymphomas. These viruses are unique in that they infect naive B cells and subsequently drive a robust polyclonal germinal center response in order to amplify the latent reservoir and to establish lifelong infection in memory B cells. The gammaherpesvirus-driven germinal center response in combination with robust infection of germinal center B cells is thought to precipitate lymphomagenesis. Importantly, host and viral factors that selectively affect the gammaherpesvirus-driven germinal center response remain poorly understood. Global deficiency of antiviral tumor-suppressive interferon regulatory factor 1 (IRF-1) selectively promotes the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and expansion of the viral latent reservoir. To determine the extent to which antiviral effects of IRF-1 are B cell intrinsic, we generated mice with conditional IRF-1 deficiency. Surprisingly, B cell-specific IRF-1 deficiency attenuated the establishment of chronic infection and the germinal center response, indicating that MHV68 may, in a B cell-intrinsic manner, usurp IRF-1 to promote the germinal center response and expansion of the latent reservoir. Further, we found that B cell-specific IRF-1 deficiency led to reduced levels of active tyrosine phosphatase SHP1, which plays a B cell-intrinsic proviral function during MHV68 infection. Finally, results of this study indicate that the antiviral functions of IRF-1 unveiled in MHV68-infected mice with global IRF-1 deficiency are mediated via IRF-1 expression by non-B cell populations.IMPORTANCE Gammaherpesviruses establish lifelong infection in over 95% of all adults and are associated with B cell lymphomas. The virus's manipulation of the germinal center response and B cell differentiation to establish lifelong infection is thought to also precipitate malignant transformation, through a mechanism that remains poorly understood. The host transcription factor IRF-1, a well-established tumor suppressor, selectively attenuates MHV68-driven germinal center response, a phenotype that we originally hypothesized to occur in a B cell-intrinsic manner. In contrast, in testing, B cell-intrinsic IRF-1 expression promoted the MHV68-driven germinal center response and the establishment of chronic infection. Our report highlights the underappreciated multifaceted role of IRF-1 in MHV68 infection and pathogenesis.

A BAFF-ling new role for NK cells in promoting marginal zone antibacterial resistance during chronic virus infection

Abstract Natural killer (NK) cells are critical for both direct control of virus replication and regulation of potentially pathogenic antiviral responses. Yet, whether NK cells also contribute to immune control of bacterial superinfections during viral infection, a significant clinical cause of morbidity and mortality, remains unknown. We find that NK cells are vital to prevent a near complete loss of B cells and macrophages associated with the marginal zone (MZ) during chronic virus infection in mice. Establishment of chronic infection in the absence of NK cells results in hyper-susceptibility to Listeria monocytogenes infection that cannot be recapitulated by NK-cell depletion prior to bacterial challenge of chronically infected mice. Therefore, we posit that preservation of MZ leukocytes by NK cells, and not direct NK cell activity, curtails post-viral susceptibility toward bacterial superinfection. Mechanistically, we show that NK cells begin expressing the crucial MZ survival factor, B-cell activating factor (BAFF), during chronic virus infection, gradually becoming a prominent source of BAFF in the spleen. In vivo blockade of BAFF in NK-sufficient mice recapitulated the infection-associated loss of MZ cells seen in NK-cell deficient mice. These results reveal a new mechanism whereby NK cells promote bacterial resistance during chronic infection by maintaining BAFF-dependent immune structures. Disruption of this activity of NK cells during certain virus infections in humans may provoke post-viral pneumonia or invasive pneumococcal disease.

Biofilm Formation and Methicillin Resistance of Staphylococcus aureus Isolated from Clinical Samples

Background: Staphylococcus aureus including methicillin resistant S. aureus (MRSA) is one of the most effective biofilm-forming organisms, biofilm contribute in protecting the microorganism from host defenses and prevent the effective penetration of antimicrobial agents. Biofilm formation is considered as an important contributing factor for the initiation and establishment of chronic infection by S. aureus and known as a major obstacle in the treatment of S. aureus infections is their ability to develop resistance to antimicrobials.
 Aims : To screen clinical Staphylococcus aureus including MRSA isolates for their biofilm forming abilities and their association with antimicrobial resistance.
 Methods: A total of 196 clinical isolates of S. aureus were obtained from different sample sources using standard microbiological techniques from three major hospitals in Gaza strip. Biofilm formation of these isolates was determined by tissue culture plate (TCP) method and tube adherence method (TM). Antimicrobial susceptibility test was performed using the modified Kirby–Bauer disk diffusion method as per Clinical and Laboratory Standards Institute guidelines. MRSA was detected using the cefoxitin disk test.
 Results: Biofilm formation was observed in 174 (88.8 %) and in 145 (74.0%) isolates of S. aureus via TCP method and TM, respectively. The highest resistance percent was for penicillin (92.9%), followed by cefoxitin (80.6%) and oxacillin (67.9%), while the lowest resistance percent was for linezolid and ceftaroline (1%). Among the 196 isolates 71.4% (N=140) were classified as MDR with a MAR index (≥ 0.2).
 A total of 158 isolates (80.6%) were identified as MRSA distributed as 90.4%, 79.4% and 70.9% from Al-Shifa, Al-Nasser pediatrics and Al-Aqsa hospitals respectively. Large proportions (82.1%) of biofilm producers were identified as MRSA. Biofilm-producing MRSA exhibited a higher percent (90.5%) when compared with the biofilm non-producer MRSA (9.5%). Importantly, 89.2% of biofilm-producing S. aureus were multidrug resistant.
 Conclusions: S. aureus isolates possessed high biofilm-forming ability. Biofilm-producing strains have very high tendency to exhibit antimicrobial resistance, multidrug resistance and methicillin resistance. Regular surveillance of biofilm formation by S. aureus and their antimicrobial resistance profile may lead more success in treating S. aureus infections.
 
 Keywords: Biofilm, MDR, MRSA, TCP, TM, Gaza strip, Palestine.

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. Its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. This review provides an overview of the viral and host factors involved in HCV entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors.