Immunodeficient Individuals Research Articles

Molecular mechanisms underlying the emergence of polygenetic antifungal drug resistance in msh2 mismatch repair mutants of Cryptococcus.

BackgroundFungal infections are common life-threatening diseases amongst immunodeficient individuals. Invasive fungal disease is commonly treated with an azole antifungal agent, resulting in selection pressure and the emergence of drug resistance. Antifungal resistance is associated with higher mortality rates and treatment failure, making the current clinical management of fungal disease very challenging. Clinical isolates from a variety of fungi have been shown to contain mutations in the MSH2 gene, encoding a component of the DNA mismatch repair pathway. Mutation of MSH2 results in an elevated mutation rate that can increase the opportunity for selectively advantageous mutations to occur, accelerating the development of antifungal resistance.ObjectivesTo characterize the molecular mechanisms causing the microevolutionary emergence of antifungal resistance in msh2 mismatch repair mutants of Cryptococcus neoformans.MethodsThe mechanisms resulting in the emergence of antifungal resistance were investigated using WGS, characterization of deletion mutants and measuring ploidy changes.ResultsThe genomes of resistant strains did not possess mutations in ERG11 or other genes of the ergosterol biosynthesis pathway. Antifungal resistance was due to small contributions from mutations in many genes. MSH2 does not directly affect ploidy changes.ConclusionsThis study provides evidence that resistance to fluconazole can evolve independently of ERG11 mutations. A common microevolutionary route to the emergence of antifungal resistance involves the accumulation of mutations that alter stress signalling, cellular efflux, membrane trafficking, epigenetic modification and aneuploidy. This complex pattern of microevolution highlights the significant challenges posed both to diagnosis and treatment of drug-resistant fungal pathogens.

Feasibility of identifying plasma donors with high measles neutralizing antibody concentrations for the use of producing a measles hyperimmune globulin for postexposure prophylaxis

Immune globulin (IG) is administered as measles postexposure prophylaxis (PEP) in people with primary immunodeficiency disorders or individuals not eligible for live virus vaccination. However, measles virus (MeV) neutralizing antibody (nAb) levels in plasma for fractionation and IG products fractionated thereof have declined. Here, the feasibility of producing a measles hyperimmune globulin (HIG) for PEP of high-risk individuals was investigated. Plasma samples (n = 384) were selected based on donor self-identification for previous MeV infection or vaccination, to determine the MeV-nAb content and compare it to the potency of plasma pools (n = 13) from the current IG manufacture. Convalescent donors have higher mean MeV-nAb concentrations (3.9 IU/mL) than vaccinated donors (2.5 IU/mL), as previously reported. However, their selection would only result in a 1.4-fold elevated nAb concentration compared to current plasma pools, which is not sufficient for HIG production. Interestingly, thirty-two donors (8%) had a MeV-nAb concentration of ≥ 8 IU/mL. The selective use of these plasma donations would result in sixfold higher plasma pool concentrations, which should permit the manufacture of the measles HIG. Further, the longitudinal analysis of a subset of individuals who repeatedly donated plasma at a high frequency revealed only a minor decline (~ 30%) of MeV-nAb levels. Repeat donations of such high-potency donors would thus facilitate the production of the measles HIG. Due to its markedly raised MeV-nAb concentration compared to standard IG, such preparation could significantly shorten infusion time and thus improve the treatment experience for both physicians and patients, especially infants.

Descriptive Study of IgG Protection for PCV13 Serotypes in Immunodeficient Patients on IVIG

Intravenous immunoglobulin (IVIG) therapy provides IgG antibodies from a donor pool to immunodeficient patients. Since PCV13 vaccine was introduced in 2010, adult donors may not have received the vaccine, thus not having PCV13 specific antibodies to donate. This descriptive analysis will highlight how pneumococcal vaccination affects Streptococcus pneumoniae protection, hospitalization rates, and associated comorbidities in immunodeficient individuals.

A discrepant presentation of bacteremia in the emergency department linked to a Fusobacterium nucleatum infection: a case report

BackgroundFusobacterium nucleatum is an anaerobic bacterium mainly responsible for acute or chronic infection of the ear, nose, and throat, potentially bacteremic with a risk of extraoral metastatic infection. Bacteremia occurs mainly in the elderly or in immunodeficient individuals, with high mortality. F. nucleatum is not the first cause of tonsillar infection in emergency departments, which are more often the consequence of a viral or streptococcal infection, but it is a risk factor for severe bacterial infection, especially in a viral pandemic context.Case presentationA 25-year-old European woman with no history presented to the emergency department with fever (38.9 °C), pharyngeal symptoms, intermittent headaches, and alteration of general condition. On examination, she presented odynophagia associated with moderate tonsillar hypertrophy, her neck was painful but flexible. A rapid diagnostic test for beta-hemolytic group streptococcus was negative. First biological analyses revealed an inflammatory syndrome with C-reactive protein of 76 mg/L. Procalcitonin was measured secondarily, and was 2.16 µg/L. Faced with discordant clinical and biological findings, a lumbar puncture was performed, which came back negative. At hour eight, hypotension was observed but corrected after filling with physiological serum. The patient was hospitalized for monitoring, based on a hypothesis of severe viral presentation. At hour 24, pyrexia confirmed this hypothesis. A spontaneous but transient improvement and no new hemodynamic event led to early discharge. At day three, she was rehospitalized for increased and continuous headaches, without hemodynamic severity. A broad-spectrum probabilistic antibiotic therapy of ceftriaxone and metronidazole was started due to first blood cultures positive for anaerobic Gram-negative bacilli, while waiting for identification of the pathogen. Three days later, F. nucleatum was identified. According to the microbiological results, antibiotic therapy was adapted with amoxicillin and clavulanic acid, and no further complications were observed during clinical or complementary examinations. The final diagnosis was a F. nucleatum oropharyngeal infection complicated by bacteremia, without metastatic spread.ConclusionThe etiologies of tonsillar infection are not limited to benign viruses or bacteria. These should not be overlooked in emergency medicine, especially when the clinical presentation is discrepant. A combination of early bacterial investigations as blood culture and close clinical monitoring is the only safe way to detect bacteremia, especially in immunocompetent patients.

A supervised U-Net based color image semantic segmentation for detection & classification of human intestinal parasites

Intestinal parasites are among the main public health problems around the world especially in underprivileged communities where overcrowded, poor environmental sanitation and lack of access for clear and safe water are prevalent. Currently, approximately 4 billion people are infected by intestinal parasites worldwide. Diseases caused by such infections can induce physical and mental disorders, and even death to children and immunodeficient individuals. Microscopy remains the gold standard in the diagnosis of human intestinal parasites. However, that manual approach requiring well-trained and experienced parasitologists is time consuming, tedious and subjective. To cope with this, machine learning tools are increasingly used to assist the medical profession in the diagnosis of intestinal parasites. Deep learning models are currently the best machine learning tools. In our work, we built a data set of 320 microscopic color images in which we detect 4 types of intestinal parasites: Ascaris Lumbricoides, Schistosoma mansoni, Trichuris Trichiura and Oxyure (or Enterobius vermicularis). Inspired by the structure of the U-Net, a deep learning model is developed for segmentation of the 4 types of intestinal parasites from the background (including fecal impurities) of the image. The proposed model has proven to be robust to underfiting and overfiting. The results show an overall detection performance of 99.8% accuracy.

A Rare Cause of Nonhealing Ulcer in an Immunocompetent Person.

Sir, A 58-year-old lady, a homemaker, presented with a nonhealing ulcer over the right lower limb for 1.5 months. There was a history of trauma following which she had developed the wound. The patient had received oral antibiotics (a combination of amoxicillin–clavulanic acid and metronidazole), but no clinical improvement was observed. There was no history suggestive of diabetes mellitus, peripheral vascular disease, varicose veins, or recurrent infections in past. There was no significant history of hospitalization before this episode. On examination, the patient was hemodynamically stable, all peripheral pulses were palpable. A 3 cm × 3 cm ulcer with sloping edges, red granulation tissue with purulent discharge on the right lateral malleolus (Gaiter's area), surrounding edema, erythema, and hyperpigmentation were seen [Figure 1].Figure 1: A 3 cm × 3 cm ulcer with sloping edges, red granulation tissue with purulent discharge on the right lateral malleolus, with surrounding edema, erythema, and pigmentationThe patient was evaluated for a nonhealing ulcer. Complete blood count was suggestive of elevated total lymphocyte count with neutrophilic predominance, with no atypical cells on the peripheral blood smear. Erythrocyte sedimentation rate 67 mm/h. Serology for HIV, HBsAg, anti-HCV antibodies was negative, and HbA1c was 5.1%. The blood culture and urine culture were sterile. Among radiological studies, a chest radiograph was normal, and the venous and arterial Dopplers of the lower limb were normal. Two-dimensional echocardiography revealed a normal study. Pus samples for acid-fast bacillus smear and culture, KOH mount, and fungal culture were also negative. Magnetic resonance imaging of the lower limb was suggestive of soft-tissue swelling and osteomyelitis of the fibular bone. Levels of immunoglobulins A, immunoglobulins M, and immunoglobulins G were 92 mg/dl, 206 mg/dl, and 976 mg/dl, respectively. Her CD4 count was 789 cells/uL. Gram stain and culture sensitivity of pus showed heavy growth of Stenotrophomonas maltophilia with sensitivity to ceftazidime, cefepime, aztreonam, and colistin. The patient was started on injection colistin, which she received for 21 days along with supportive measures. By day 14, there was a gross improvement with complete healing of the wound in a month. S. maltophilia is a gram-negative bacillus with intrinsic drug resistance to various antimicrobials including beta-lactams, fluoroquinolones, and aminoglycosides.[1] It is known to cause a variety of infections in immunocompromised individuals, especially candidates with hematopoietic stem-cell transplantation, receiving cytotoxic chemotherapy, and those with neutropenia.[1] It commonly causes infection in immunodeficient individuals; however, in recent times, a surge in the number of cases of immunocompetent individuals developing skin and soft-tissue infection has been observed. Our patient acquired infection of S. maltophilia probably from the environment despite being immunocompetent.[2] Since the patient showed a dramatic response to culture-guided antibiotics with complete healing of the wound, the possibility of colonization was less likely. Therefore, we conclude that a possibility of S. maltophilia should also be kept as a possibility in cases of nonhealing ulcers even in immunocompetent individuals. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed. Research quality and ethics statement The authors followed applicable EQUATOR Network (http://www.equator-network.org/) guidelines, notably the CARE guideline, during the conduct of this report. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Proteome-wide prediction and analysis of the Cryptosporidium parvum protein–protein interaction network through integrative methods

As one of the most studied Apicomplexan parasite Cryptosporidium, Cryptosporidium parvum (C. parvum) causes worldwide serious diarrhea disease cryptosporidiosis, which can be deadly to immunodeficiency individuals, newly born children, and animals. Proteome-wide identification of protein–protein interactions (PPIs) has proven valuable in the systematic understanding of the genome-phenome relationship. However, the PPIs of C. parvum are largely unknown because of the limited experimental studies carried out. Therefore, we took full advantage of three bioinformatics methods, i.e., interolog mapping (IM), domain-domain interaction (DDI)-based inference, and machine learning (ML) method, to jointly predict PPIs of C. parvum. Due to the lack of experimental PPIs of C. parvum, we used the PPI data of Plasmodium falciparum (P. falciparum), which owned the largest number of PPIs in Apicomplexa, to train an ML model to infer C. parvum PPIs. We utilized consistent results of these three methods as the predicted high-confidence PPI network, which contains 4,578 PPIs covering 554 proteins. To further explore the biological significance of the constructed PPI network, we also conducted essential network and protein functional analysis, mainly focusing on hub proteins and functional modules. We anticipate the constructed PPI network can become an important data resource to accelerate the functional genomics studies of C. parvum as well as offer new hints to the target discovery in developing drugs/vaccines.

Role of metapneumoviral glycoproteins in the evasion of the host cell innate immune response.

Human metapneumovirus (HMPV), an unsegmented negative-strand RNA virus, is the second most detected respiratory pathogen and one of the leading causes of respiratory illness in infants and immunodeficient individuals. HMPV infection of permissive cells in culture triggers a transient IFN response, which is efficiently suppressed later in infection. We report that two structural glycoproteins of the virus - namely G (Glycoprotein) and SH (Small Hydrophobic) - suppress the type I interferon (IFN) response in cell culture. This is manifested by inhibition of diverse steps of IFN induction and response, such as phosphorylation and nuclear translocation of IFN regulatory factor-3 and -7 (IRF3, IRF7), major transcription factors of the IFN gene. Furthermore, HMPV suppresses the cellular response to IFN by inhibiting the phosphorylation of STAT1 (Signal Transducer and Activator of Transcription 1), required for the induction of IFN-stimulated genes that act as antivirals. Site-directed mutagenesis revealed an important role of critical cysteine (Cys) residues in the Cys-rich carboxy terminal region of the SH protein in IFN suppression, whereas for G, the ectodomain plays a role. These results shed light on the mechanism of IFN suppression by HMPV, and may also offer avenues for new antiviral approaches in the future.

Fpr2/CXCL1/2 Controls Rapid Neutrophil Infiltration to Inhibit Streptococcus agalactiae Infection.

Streptococcus agalactiae, also known as group B streptococcus (GBS), can cause pneumonia, meningitis, and bacteremia, making it a pathogen that can increase the risk of death in newborns and immunodeficient individuals. Neutrophils are the first barrier to a host’s innate immune defense against these infections. Fpr2(Formyl peptide receptor 2) is an important chemotactic receptor of neutrophils, though its activation would cause pro- and anti-inflammatory effects. In this study, we found that mice without Fpr2 receptor were highly susceptible to GBS infections. These mice demonstrated decreased chemotaxis to neutrophils, decreased bactericidal ability of neutrophils, and high mortality. RNA-seq and Luminex assay indicated that Fpr2 activates key signal molecules downstream and produces chemokines CXCL1/2 to chemotaxis neutrophils. Like Fpr2-/-, CXCL1/2 or neutrophil depletion impairs host’s ability to defend against GBS infection. Altogether, these data indicate that Fpr2 contributes to a host’s ability to control GBS infection and that a lack of Fpr2 was associated with selective impairment during the production of chemokines CXCL1 and CXCL2 as well as neutrophil recruitment. Here, We clarified that Fpr2, as a chemotactic receptor, could not only directly chemotactic neutrophils, but also regulate the production of chemokines to control infection by chemotactic neutrophils.

Efficacy of Lactobacillus fermentum Isolated from the Vagina of a Healthy Woman against Carbapenem-Resistant Klebsiella Infections In Vivo.

Carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase are increasingly reported worldwide and have become more and more resistant to nearly all antibiotics during the past decade. The emergence of K. pneumoniae strains with decreased susceptibility to carbapenems, which are used as a last resort treatment option, is a significant threat to hospitalized patients worldwide as K. pneumoniae infection is responsible for a high mortality rate in the elderly and immunodeficient individuals. This study used Lactobacillus fermentum as a candidate probiotic for treating CRE-related infections and investigated its effectiveness. We treated mice with L. fermentum originating from the vaginal fluid of a healthy Korean woman and evaluated the Lactobacilli's efficacy in preventive, treatment, non-establishment, and colonization mouse model experiments. Compared to the control, pre-treatment with L. fermentum significantly reduced body weight loss in the mouse models, and all mice survived until the end of the study. The oral administration of L. fermentum after carbapenemresistant Klebsiella (CRK) infection decreased mortality and illness severity during a 2-week observation period and showed that it affects other strains of CRK bacteria. Also, the number of Klebsiella bacteria was decreased to below 5.5 log10 CFU/ml following oral administration of L. fermentum in the colonization model. These findings demonstrate L. fermentum's antibacterial activity and its potential to treat CRE infection in the future.

A case of ocular neurosyphilis in a patient with HIV

A case of ocular neurosyphilis in a patient with HIV

PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice

While Epstein–Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

Mucormycosis: A manifestation in COVID-19 infection

Patients with COVID-19 may develop higher susceptibility to fungal co-infections. Mucormycosis is a rare and often life-threatening disease caused by vascular invasion by hyphae, leading to thrombosis and necrosis. Mucormycosis has been the cause of morbidity and mortality in transplant, ICU and immunodeficient individuals over a long period of time, but in India, a rapid increase in the number of cases seen in unexpected patients with Covid-19, which is a major concern. Covid-19 patients, already immunocompromised with underlying condition, had been exposed to severe Covid-19 infection and been using steroids, were at greater risk of developing mucormycosis. Out of ten cases reported by Sir Ganga Ram Hospital, Delhi India in December2020, five succumbed to mucormycosis. Another unique feature reported in mucor patients was age, most were elderly with ages between 45 to 60 years. The mortality rate is currently estimated at 50% in India. Symptoms such as nasal congestion, swelling of the eye or cheeks and dark brown crusts inside the nose should be reported immediately and anti-fungal treatment should be started.

Ethical Challenges in Mandatory Vaccination Against Covid-19

"The current COVID-19 pandemic creates unique and unmatched challenges to healthcare system worldwide. For vulnerable hosts such as immunodeficient individuals, older people, and those with additional comorbidities, SARS-CoV-2 infection primary raises high risk of death outcome. The population should be protected from the further distribution of SARS-CoV-2 through contact distancing, the using of protective masks and other hygiene measures, but also through vaccination, since vaccinations are one of the most important primary prevention tools. Therefore, vaccine is anticipated to protect the vaccinated person from severe disease if infected by the specific pathogen the vaccine was targeted against. On 11th March 2020 the World Health Organization (WHO) declared COVID-19 as pandemic, yet in 2019, the vaccine hesitancy was named by the WHO as one of the top 10 threats to global health. Acceptance of the vaccine is critical to its success and with the rise of the anti-vaxxer movement; continuous increasing public awareness is needed without any delay. Mandatory vaccination, including for COVID-19, could be ethically justified if the threat to public health is serious, the confidence in efficacy and safety is high, the anticipated utility of mandatory vaccination is superior to the alternatives, but also if the penalties or charges for non-compliance are balanced. Besides, one can argue that vaccination isn’t just an individual preference, namely vaccination protects those who can’t be vaccinated. So, although vaccine mandates for adults may be legal, so far they have been applied restrictively to select groups, such as health care providers, but also in businesses that require in-person attendance, education-related sectors, or in long-distance travel businesses, rather widely enforced. As for the vaccination hesitancy, the results of majority of studies indicated vaccine safety, efficacy, protection duration, and potential side effects as the most important reasons for COVID-19 vaccination hesitancy. Apart from vaccine-specific concerns, reasons for vaccine hesitancy also included a need for more information, antivaccine attitudes or beliefs, and a lack of trust. Taking into account the need to keep the world population over the herd immunity threshold, a continuous action is going to be expected from public health authorities to maintain trust and reduce COVID-19 vaccine hesitancy. "

Some Indicators of Nonspecific Immunity of Various Breeding Groups of Carp in Different Periods of the Annual Cycle

A comparative study of some parameters of nonspecific immunity in the Angelinskii rubella-resistant variant of the scaly and mirror groups of carp in the prespawning and late feeding periods was carried out. The level of the antimicrobial properties of the C-reactive protein, nonspecific immune complexes in the blood serum, and phagocytic activity of neutrophils were studied. The study showed interbreed and inter-seasonal differences in some indicators. It is concluded that the higher level of bacteriostatic activity of blood serum in the rubella-resistant variant, compared with other groups of carp, provides a low percentage of immunodeficient individuals and resistance to pathogens of infectious diseases.

Oral health's inextricable connection to systemic health: Special populations bring to bear multimodal relationships and factors connecting periodontal disease to systemic diseases and conditions.

The landscape in dentistry is changing as emerging studies continue to reveal that periodontal health impacts systemic health, and vice versa. Population studies, clinical studies, and in vitro animal studies underscore the critical importance of oral health to systemic health. These inextricable relationships come to the forefront as oral diseases, such as periodontal disease, take root. Special populations bring to bear the multimodal relationships between oral and systemic health. Specifically, periodontal disease has been associated with diabetes, metabolic syndrome, obesity, eating disorders, liver disease, cardiovascular disease, Alzheimer disease, rheumatoid arthritis, adverse pregnancy outcomes, and cancer. Although bidirectional relationships are recognized, the potential for multiple comorbidities, relationships, and connections (multimodal relationships) also exists. Proposed mechanisms that mediate this connection between oral and systemic health include predisposing and precipitating factors, such as genetic factors (gene polymorphisms), environmental factors (stress, habits—such as smoking and high‐fat diets/consumption of highly processed foods), medications, microbial dysbiosis and bacteremias/viremias/microbemias, and an altered host immune response. Thus, in a susceptible host, these predisposing and precipitating factors trigger the onset of periodontal disease and systemic disease/conditions. Further, high‐throughput sequencing technologies are shedding light on the dark matter that comprises the oral microbiome. This has resulted in better characterization of the oral microbial dysbiosis, including putative bacterial periodontopathogens and shifts in oral virome composition during disease. Multiple laboratory and clinical studies have illustrated that both eukaryotic and prokaryotic viruses within subgingival plaque and periodontal tissues affect periodontal inflammation, putative periodontopathogens, and the host immune response. Although the association between herpesviruses and periodontitis and the degree to which these viruses directly aggravate periodontal tissue damage remain unclear, the benefits to periodontal health found from prolonged administration of antivirals in immunocompromised or immunodeficient individuals demonstrates that specific populations are possibly more susceptible to viral periodontopathogens. Thus, it may be important to further examine the implications of viral pathogen involvement in periodontitis and perhaps it is time to embrace the viral dark matter within the periodontal environment to fully comprehend the pathogenesis and systemic implications of periodontitis. Emerging data from the coronavirus disease 2019 pandemic further underscores the inextricable connection between oral and systemic health, with high levels of the severe acute respiratory syndrome coronavirus 2 angiotensin‐converting enzyme 2 receptor noted on oral tissues (tongue) and an allostatic load or overload paradigm of chronic stress likely contributing to rapid breakdown of oral/dental, periodontal, and peri‐implant tissues. These associations exist within a framework of viremias/bacteremias/microbemias, systemic inflammation, and/or disturbances of the immune system in a susceptible host. A thorough review of systemic and oral diseases and conditions and their mechanistic, predisposing, and precipitating factors are paramount to better addressing the oral and systemic health and needs of our patients.

Biological contamination due to microsporidia in urban soils: an emerging risk?

BACKGROUND AND AIM: Topsoils can be a source of emerging opportunistic and zoonotic parasites. Children and immunodeficient individuals can be accidentally exposed to microsporidia spores when spending time in urban parks and recreational areas. The aim of this study was to determine the presence and distribution of human-related microsporidian species in urban/industrial areas in Alcalá de Henares (Madrid Region, Spain). METHODS: 227 topsoil samples were collected July 2017 across different urban (n=155) and industrial (n=60) areas in Alcalá, and in a public garden in the city centre (n=12). DNA was extracted from concentrated pellet by disrupting the spores using Fast-Prep for Soil, followed by SYBR Green real-time PCR for simultaneous detection of {Enterocytozoon bieneusi} and {Encephalitozoon} spp. ({E. intestinalis}, {E. hellem} and {E. cuniculi}). RESULTS:Real-time amplification with MsRTf1/MsRTr1 primer set and differential melting curve analysis confirmed presence of {E. bieneusi} (n=9; 5 urban, 4 industrial), and {Encephalitozoon} spp. in 22 of the soil samples: three {E. cuniculi} (2 urban, 1 industrial); ten {E. intestinalis} (8 urban, 2 industrial); nine {E. intestinalis}/{E. hellem} (6 urban, 3 industrial). Microsporidia spores were not found in any of the samples monitored in public gardens. Urban animals have been suggested to play a role in the distribution of these emerging parasites through their droppings. Thus, similar studies carried out in Madrid city have detected microsporidia in animal faeces collected from public parks but not in their soils, which highlights the significance of our study. CONCLUSIONS:Our results indicate a moderate prevalence and distribution of human-related microsporidia in soils in Alcalá, which could represent a public health concern. Interventions to protect the public might be necessary as the presence/distribution of these biothreats could be higher, as potential false-negative results due to low DNA parasite concentration and/or the presence of environmental PCR inhibitors should be considered. KEYWORDS: Microsporidia, urban topsoils, human risks, biological contamination

Intronic SNP - rs9288953 Of Suppressor Molecule Btla Confers Susceptibility To B-Cell Chronic Lymphocytic Leukemia

The contribution of the immune system to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) have been receiving growing attention. The increase of cancer risk in immunodeficient individuals has been documented in population and experimental studies (Gurlich et al 2007; Swann et al 2007, Smyth et al 2006). A new member of co-inhibitory molecules B - and T-lymphocyte attenuator (BTLA) that inhibits lymphocyte activation could influence cancer susceptibility (Watanabe et al 2003). A high expression of BTLA was observed on malignant B cells from chronic lymphocytic leukemia/small lymphocytic leukemia (M'Hidi et al. 2009). As BTLA synthesis depends on the rate of gene transcription and/or translation, polymorphism in the corresponding gene might result in abnormal expression and function.The aim of this studywas to evaluate possible associations between selected on the basis of literature (Fu et al 2009) single nucleotide polymorphisms (SNPs) existing within exons and introns of BTLA gene: rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T and susceptibility to B-CLL.Genotyping was done using allelic discrimination method with the TaqMan SNP Genotyping Assays in 300 B-CLL patients (pts.) (203 pts. from Department of Hematology, Medical University Lodz, Poland and 97 from Department of Clinical Immunology and Immunotherapy, Medical University of Lublin, Poland (138 female/162 male) and in 417 healthy individuals (168 female/243 male) originating from the same region of Poland.The significant differences in distribution of alleles and genotypes was observed for intronic SNP -rs9288953. The frequency of T allele and T alleles carriers (CT+TT genotypes) were higher in patients than in healthy controls as compare to C allele and CC genotypes (0.458 vs. 0.375, p=0.0016; OR:1.41, 95%CI - 1.13-1.75 and 0.677 vs. 0.606, p=0.0022; OR:1.67, 95%CI- 1.16-1.2.40, respectively). None of other investigated SNPs have shown different frequency of alleles or genotypes in group of patients and controls.The haplotype analysis of rs9288952A>G, rs1844089G>A, rs2705535C>T and 9288953C>T showed that the most common haplotypes rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[C] (named as AGCC) and rs9288952A>G[A]/rs1844089G>A[G]/rs2705535C>T[C]/ 9288953C>T[T] (named as AGCT) which were differentiate only by variants of 9288953C>T SNP were completely contrary associated with disease susceptibility. Haplotype AGCC is associated with decreased risk of B-CLL (OR: 0.72, 95%CI-0.59-0.90 p with Bonferroni correction=0.01), while AGCT with increased risk of disease (OR: 1.42, 95%CI-1.14-1.76 p with Bonferroni correction=0.005)Our data indicated for the first time the possible role of intronic polymorphism 9288953C>T in gene encoding suppressor molecule BTLA in susceptibility to B-CLL. ReferencesFu Z, Li D, Jiang W, Wang L, Zhang J, Xu F, Pang D, Li D. Association of BTLA gene polymorphisms with the risk of malignant breast cancer in Chinese women of Heilongjiang Province. Breast Cancer Res Treat. 2010 Feb;120(1):195-202.Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet. 2007 Jul 7;370(9581):59-67.M'Hidi H, Thibult ML, Chetaille B, Rey F, Bouadallah R, Nicollas R, Olive D, Xerri L. High expression of the inhibitory receptor BTLA in T-follicular helper cells and in B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia.Am J Clin Pathol. 2009 Oct;132(4):589-96.Smyth MJ, Dunn GP, Schreiber RD. Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol. 2006;90:1-50.Swann JB, Smyth MJ. Immune surveillance of tumors. J Clin Invest. 2007 May;117(5):1137-46.Watanabe N, Gavrieli M, Sedy JR, Yang J, Fallarino F, Loftin SK, Hurchla MA, Zimmerman N, Sim J, Zang X, Murphy TL, Russell JH, Allison JP, Murphy KM. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.Nat Immunol. 2003 Jul;4(7):670-9. Disclosures:No relevant conflicts of interest to declare.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ Tcells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir

Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.