Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

Intratumoral stem-like CCR4+ regulatory T cells orchestrate the immunosuppressive microenvironment in HCC associated with hepatitis B.

BACKGROUND & AIMS: Regulatory T cell (Treg) depletion increases antitumor immunity. However, severe autoimmunity can occur following systemic loss of Tregs, which could be avoided by selectively depleting intratumoral Tregs. Herein, we aimed to investigate the role of tumor-infiltrating CCR4+ Tregs in hepatocellular carcinoma (HCC) and to provide a potential target strategy for immunotherapy. METHODS: CCR4+ Tregs were analyzed by flow cytometry in murine models and clinical samples. The function of tumor-infiltrating and induced CCR4+ Tregs was interrogated by genetic and epigenetic approaches. To block CCR4+ Treg chemotaxis, we developed an N-terminus recombinant protein of CCR4 (N-CCR4-Fc) as a neutralizing pseudo-receptor that effectively bound to its ligand CCL22. The efficacy of CCR4 antagonism as an immunotherapeutic agent was evaluated by tumor weights, growth kinetics and survival curves. RESULTS: CCR4+ Tregs were the predominant type of Tregs recruited to hepatitis B-associated HCC (HBV+ HCC), correlating with sorafenib resistance and HBV load titers. Compared with CCR4- Tregs, CCR4+ Tregs exhibited increased IL-10 and IL-35 expression, and enhanced functionality in suppressing CD8+ T cells. CCR4+ Tregs also displayed PD-1+TCF1+ stem-like properties. ATAC-seq data revealed substantial chromatin remodeling between tumor-infiltrating Tregs (TIL-Tregs) and induced Tregs, suggesting that long-term chromatin reprogramming accounted for the acquisition of enhanced immunosuppressive stem-like specificity by CCR4+ TIL-Tregs. Treatment with a CCR4 antagonist or N-CCR4-Fc blocked intratumoral Treg accumulation, overcame sorafenib resistance, and sensitized tumors to PD-1 checkpoint blockade. CONCLUSIONS: Intratumoral stem-like CCR4+ Tregs orchestrated immunosuppressive resource cells in the tumor microenvironment. CCR4 could be targeted to enhance antitumor immunity by specifically blocking infiltration of Tregs into the tumor microenvironment and inhibiting maintenance of the TIL-Treg pool. LAY SUMMARY: Targeting regulatory T cells is a promising approach in cancer immunotherapy; however, severe autoimmunity can occur following systemic regulatory T cell loss. This could be avoided by selectively depleting intratumoral regulatory T cells. Herein, targeting intratumoral stem-like CCR4+ regulatory T cells helped to overcome sorafenib resistance and sensitize tumors to immune checkpoint blockade in mouse models of liver cancer. This approach could have wide clinical applicability.

Viral Enteritis in Solid-Organ Transplantation.

Solid organ transplant recipients are at increased risk for infections due to chronic immunosuppression. Diarrhea is a commonly encountered problem post transplantation, with infectious causes of diarrhea being a frequent complication. Viral infections/enteritides in solid organ transplant recipients often result from frequently encountered pathogens in this population such as cytomegalovirus, adenovirus, and norovirus. However, several emerging viral pathogens are increasingly being recognized as more sensitive diagnostic techniques become available. Treatment is often limited to supportive care and reduction in immunosuppression, though antiviral therapies mayplay a role in the treatment in certain diseases. Viral enteritis is an important entity that contributes to morbidity and mortality in transplant recipients.

In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host.

Zoonotic transfer of animal pathogens to human hosts can generate novel agents, but the genetic events following such host jumps are not well studied. Here we characterize the mechanisms driving adaptive evolution of the emerging zoonotic pathogen Bordetella hinzii in a patient with interleukin-12 receptor ß1 deficiency. Genomic sequencing of 24 B. hinzii isolates cultured from blood and stool over 45 months revealed a clonal lineage that had undergone extensive within-host genetic and phenotypic diversification. Twenty of 24 isolates shared an E9G substitution in the DNA polymerase III ε-subunit active site, resulting in a proofreading deficiency. Within this proofreading-deficient clade, multiple lineages with mutations in DNA repair genes and altered mutational spectra emerged and dominated clinical cultures for more than 12 months. Multiple enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways were repeatedly mutated, suggesting rapid metabolic adaptation to the human environment. Furthermore, an excess of G:C > T:A transversions suggested that oxidative stress shaped genetic diversification during adaptation. We propose that inactivation of DNA proofreading activity in combination with prolonged, but sub-lethal, oxidative attack resulting from the underlying host immunodeficiency facilitated rapid genomic adaptation. These findings suggest a fundamental role for host immune phenotype in shaping pathogen evolution following zoonotic infection.

COVID-19 Severity Potentially Modulated by Cardiovascular-Disease-Associated Immune Dysregulation.

Patients with underlying cardiovascular conditions are particularly vulnerable to severe COVID-19. In this project, we aimed to characterize similarities in dysregulated immune pathways between COVID-19 patients and patients with cardiomyopathy, venous thromboembolism (VTE), or coronary artery disease (CAD). We hypothesized that these similarly dysregulated pathways may be critical to how cardiovascular diseases (CVDs) exacerbate COVID-19. To evaluate immune dysregulation in different diseases, we used four separate datasets, including RNA-sequencing data from human left ventricular cardiac muscle samples of patients with dilated or ischemic cardiomyopathy and healthy controls; RNA-sequencing data of whole blood samples from patients with single or recurrent event VTE and healthy controls; RNA-sequencing data of human peripheral blood mononuclear cells (PBMCs) from patients with and without obstructive CAD; and RNA-sequencing data of platelets from COVID-19 subjects and healthy controls. We found similar immune dysregulation profiles between patients with CVDs and COVID-19 patients. Interestingly, cardiomyopathy patients display the most similar immune landscape to COVID-19 patients. Additionally, COVID-19 patients experience greater upregulation of cytokine- and inflammasome-related genes than patients with CVDs. In all, patients with CVDs have a significant overlap of cytokine- and inflammasome-related gene expression profiles with that of COVID-19 patients, possibly explaining their greater vulnerability to severe COVID-19.

Transcription of human papillomaviruses in nonmelanoma skin cancers of the immunosuppressed.

Nonmelanoma skin cancer (NMSC) has a greatly increased incidence among the immunosuppressed and the DNA of human papillomavirus (HPV) is commonly found in these tumors. To investigate if there are any actively transcribed HPV infections in these tumors, we identified all skin cancers diagnosed after solid organ transplantation in Sweden during 1964-2011 (n = 7614 NMSCs) and requested the diagnostic tumor blocks from the corresponding pathology archives. For the present study, we selected diagnostic specimens from 345 NMSC and performed whole genome transcriptome analysis using NovaSeq (Illumina), in comparison with three cervical cancers. Although we obtained an abundance of high-quality paired reads per sample (median of 35 million reads), only 15 NMSC specimens contained HPV transcription. Three specimens had transcription of oncogenic anogenital HPVs (HPV16 and 56), six tumors had transcription of HPVs from the beta-2 species (three HPV38, two with HPV23 and one with HPV107) and then there was one observation each of transcription of HPVs 3, 26, 57, 147, 158, 168 and of two nonestablished HPV types belonging to the gamma genus. In conclusion, transcription of specific HPV types can be found in NMSC among the immunosuppressed, but this is not common.

Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source.

NBSGW mice are highly immunodeficient and carry a hypomorphic mutation in the c-kit gene, providing a host environment that supports robust human hematopoietic expansion without pre-conditioning. These mice thus provide a model to investigate human hematopoietic engraftment in the absence of conditioning-associated damage. We compared transplantation of human CD34+ HSPCs purified from three different sources: umbilical cord blood, adult bone marrow, and adult G-CSF mobilized peripheral blood. HSPCs from mobilized peripheral blood were significantly more efficient (as a function of starting HSPC dose) than either cord blood or bone marrow HSPCs at generating high levels of human chimerism in the murine blood and bone marrow by 12 weeks post-transplantation. While T cells do not develop in this model due to thymic atrophy, all three HSPC sources generated a human compartment that included B lymphocytic, myeloid, and granulocytic lineages. However, the proportions of these lineages varied significantly according to HSPC source. Mobilized blood HSPCs produced a strikingly higher proportion of granulocyte lineage cells (~35% as compared to ~5%), whereas bone marrow HSPC output was dominated by B lymphocytic cells, and cord blood HSPC output was enriched for myeloid lineages. Following transplantation, all three HSPC sources showed a shift in the CD34+ subset towards CD45RA+ progenitors along with a complete loss of the CD45RA-CD49f+ long-term HSC subpopulation, suggesting this model promotes mainly short-term HSC activity. Mice transplanted with cord blood HSPCs maintained a diversified human immune compartment for at least 36 weeks after the primary transplant, although mice given adult bone marrow HSPCs had lost diversity and contained only myeloid cells by this time point. Finally, to assess the impact of non-HSPCs on transplantation outcome, we also tested mice transplanted with total or T cell-depleted adult bone marrow mononuclear cells. Total bone marrow mononuclear cell transplants produced significantly lower human chimerism compared to purified HSPCs, and T-depletion rescued B cell levels but not other lineages. Together these results reveal marked differences in engraftment efficiency and lineage commitment according to HSPC source and suggest that T cells and other non-HSPC populations affect lineage output even in the absence of conditioning-associated inflammation.

Multidrug-Resistant Gram-Negative Bacteria Decolonization in Immunocompromised Patients: A Focus on Fecal Microbiota Transplantation.

Antimicrobial resistance is an important issue for global health; in immunocompromised patients, such as solid organ and hematological transplant recipients, it poses an even bigger threat. Colonization by multidrug-resistant (MDR) bacteria was acknowledged as a strong risk factor to subsequent infections, especially in individuals with a compromised immune system. A growing pile of studies has linked the imbalance caused by the dominance of certain taxa populating the gut, also known as intestinal microbiota dysbiosis, to an increased risk of MDR bacteria colonization. Several attempts were proposed to modulate the gut microbiota. Particularly, fecal microbiota transplantation (FMT) was successfully applied to treat conditions like Clostridioides difficile infection and other diseases linked to gut microbiota dysbiosis. In this review we aimed to provide a look at the data gathered so far on FMT, focusing on its possible role in treating MDR colonization in the setting of immunocompromised patients and analyzing its efficacy and safety.

Microevolution of Campylobacter jejuni during long-term infection in an immunocompromised host.

Campylobacteriosis typically manifests as a short-lived, self-limiting gastrointestinal infection in humans, however prolonged infection can be seen in cases with underlying immunodeficiency. Public Health England received 25 isolates of Campylobacter jejuni from an individual with combined variable immunodeficiency over a period of 15 years. All isolates were typed and archived at the time of receipt. Whole genome sequencing (WGS) and antimicrobial susceptibility testing were performed to examine the relatedness of the isolates and to investigate the changes in the genome that had taken place over the course of the infection. Genomic typing methods were compared to conventional phenotypic methods, and revealed that the infection was caused by a single, persistent strain of C. jejuni belonging to clonal complex ST-45, with evidence of adaptation and selection in the genome over the course of the infection. Genomic analysis of sequence variants associated with antimicrobial resistance identified the genetic background behind rRNA gene mutations causing variable levels of resistance to erythromycin. This application of WGS to examine a persistent case of campylobacteriosis provides insight into the mutations and selective pressures occurring over the course of an infection, some of which have important clinical relevance.

Rapid and precise diagnosis of T. marneffei pulmonary infection in a HIV-negative patient with autosomal-dominant STAT3 mutation: a case report.

BACKGROUND: Talaromyces marneffei, also named Penicillium marneffei, is an opportunistic pathogen that can cause systemic or limited infection in human beings. This infection is especially common in human immunodeficiency virus (HIV)-infected hosts; however, it has also been recently reported in HIV-negative hosts. Here, we report a very rarely seen case of T. marneffei pulmonary infection in a non-HIV-infected patient with signal transducer and activator of transcription 3 (STAT3) mutation. CASE PRESENTATION: A 34-year-old woman was admitted to our hospital for uncontrollable nonproductive cough and dyspnea with exercise. She had been immunocompromised since infancy. Computerized tomography scan showed multiple ground glass opacities with multiple bullae in both lungs. Next generation sequencing (NGS) of the bronchoalveolar lavage fluid identified T. marneffei nucleotide sequences. Culture of bronchoscopy specimens further verified the results. The patient was HIV negative, and blood gene detection indicated STAT3 mutation. To date, following the application of itraconazole, the patient has recovered satisfactorily. CONCLUSION: In clinical practice, T. marneffei infection among HIV-negative individuals is relatively rare, and we found that patients who are congenitally immunocompromised due to STAT3 mutation may be potential hosts. Early diagnosis and timely treatment are expected to improve the prognosis of T. marneffei infection. NGS is a powerful technique that may play an important role in this progress. The reviews of this paper are available via the supplemental material section.

Incidence of hepatitis B virus infection among human immunodeficiency virus-infected treatment naïve adults in Botswana.

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection is highest in sub-Saharan Africa and results in accelerated clinical outcomes compared with HBV or HIV mono-infection. HBV clearance rates are higher in healthy adults; however, in sub-Saharan Africa, there are limited data on clearance of incident HBV in HIV-infected adults. Therefore, we sought to estimate HBV incidence and HBV surface antigen (HBsAg) clearance in HIV-infected adults in Botswana.This was a retrospective longitudinal study of 442 HIV-1C infected treatment naïve patients enrolled in a previous Botswana Harvard AIDS Institute Partnership study. Archived plasma samples from 435 HIV-infected treatment naïve participants were screened for HBsAg and HBV core antibody (anti-HBc). HBsAg was evaluated annually over a 4-year period, and HBV deoxyribonucleic acid (DNA) levels of HBsAg-positive chronic and incident patients were quantified.Baseline median CD4+ T-cell count was 458 cells/µL [Q1, Q3: 373, 593], and median HIV viral load was 4.15 copies/mL [Q1, Q3: 3.46, 4.64]. Twenty two HBV incident cases occurred, representing an incidence of 3.6/100 person-years [95% CI: 2.2-5.6]. All incident HBV cases with a follow-up sample available for screening (13/22) cleared HBsAg. Detectable HBV viral loads among chronic and incident cases ranged between 5.15 × 10 to 1.4 × 10 IU/L and 1.80 × 10 to 1.7 × 10 IU/mL, respectively.We report high HBV incidence associated with elevated HBV DNA levels despite high CD4+ T-cell counts in HIV-infected patients in Botswana. These incidence cases represent a potential source of HBV transmission in the population. Scaling-up of HIV treatment strategies utilizing antiretroviral therapy regimens with anti-HBV activity coupled with screening for HBV infections in households of the HBsAg-positive cases is recommended.

Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine.

The humanization of animals is a powerful tool for the exploration of human disease pathogenesis in biomedical research, as well as for the development of therapeutic interventions with enhanced translational potential. Humanized models enable us to overcome biologic differences that exist between humans and other species, while giving us a platform to study human processes in vivo. To become humanized, an immune-deficient recipient is engrafted with cells, tissues, or organoids. The mouse is the most well studied of these hosts, with a variety of immunodeficient strains available for various specific uses. More recently, efforts have turned to the humanization of other animal species such as the rat, which offers some technical and immunologic advantages over mice. These advances, together with ongoing developments in the incorporation of human transgenes and additional mutations in humanized mouse models, have expanded our opportunities to replicate aspects of human allotransplantation and to assist in the development of immunotherapies. In this review, the immune and tissue humanization of various species is presented with an emphasis on their potential for use as models for allotransplantation, graft versus host disease, and regenerative medicine.

Evaluation of Genotypic Antiviral Resistance Testing as an Alternative to Phenotypic Testing in a Patient with DOCK8 Deficiency and Severe HSV-1 Disease.

Antiviral resistance frequently complicates the treatment of herpes simplex virus (HSV) infections in immunocompromised patients. Here we present the case of an adolescent boy with dedicator of cytokinesis 8 (DOCK8) deficiency, who experienced recurrent infections with resistant HSV-1. We used both phenotypic and genotypic methodologies to characterize the resistance profile of HSV-1 in the patient and conclude that genotypic testing outperformed phenotypic testing. We also present the first analysis of intrahost HSV-1 evolution in an immunocompromised patient. While HSV-1 can remain static in an immunocompetent individual for decades, the virus from this patient rapidly acquired genetic changes throughout its genome. Finally, we document a likely case of transmitted resistance in HSV-1 between the patient and his brother, who also has DOCK8 deficiency. This event demonstrates that resistant HSV-1 is transmissible among immunocompromised persons.

ILC3-derived OX40L is essential for homeostasis of intestinal Tregs in immunodeficient mice.

OX40L is one of the co-stimulatory molecules that can be expressed by splenic lymphoid tissue inducer (Lti) cells, a subset of group 3 innate lymphoid cells (ILC3s). OX40L expression in subsets of intestinal ILC3s and the molecular regulation of OX40L expression in ILC3s are unknown. Here, we showed intestinal ILC3s marked as an OX40Lhigh population among all the intestinal leukocytes and were the dominant source of OX40L in Rag1-/- mice. All ILC3 subsets expressed OX40L, and NCR-ILC3s were the most abundant source of OX40L. The expression of OX40L in ILC3s could be upregulated during inflammation. In addition to tumor necrosis factor (TNF)-like cytokine 1A (TL1A), which has been known as a trigger for OX40L, we found that Poly (I:C) representing viral stimulus promoted OX40L expression in ILC3s via a cell-autonomous manner. Furthermore, we demonstrated that IL-7-STAT5 signaling sustained OX40L expression by ILC3s. Intestinal regulatory T cells (Tregs), most of which expressed OX40, had defective expansion in chimeric mice, in which ILC3s were specifically deficient for OX40L expression. Consistently, co-localization of Tregs and ILC3s was found in the cryptopatches of the intestine, which suggests the close interaction between ILC3s and Tregs. Our study has unveiled the crosstalk between Tregs and ILC3s in mucosal tissues through OX40-OX40L signaling, which is crucial for the homeostasis of intestinal Tregs.

Recurrent Herpes Zoster Ophthalmicus in a Patient With a Novel Toll-Like Receptor 3 Variant Linked to Compromised Activation Capacity in Fibroblasts.

BACKGROUND: Herpes zoster ophthalmicus occurs primarily in elderly or immunocompromised individuals after reactivation of varicella zoster virus (VZV). Recurrences of zoster ophthalmicus are uncommon because the reactivation efficiently boosts anti-VZV immunity. A 28-year-old female presented to our clinic with a history of multiple recurrences of zoster ophthalmicus. METHODS: Whole-exome sequencing (WES), analyses of VZV T-cell immunity, and pathogen recognition receptor function in primary antigen-presenting cells (APCs) and fibroblasts were performed. RESULTS: Normal VZV-specific T-cell immunity and antibody response were detected. Whole-exome sequencing identified a heterozygous nonsynonymous variant (c.2324C > T) in the Toll-like receptor 3 (TLR3) gene resulting in formation of a premature stop-codon. This alteration could potentially undermine TLR3 signaling in a dominant-negative fashion. Therefore, we investigated TLR3 signaling responses in APCs and fibroblasts from the patient. The APCs responded efficiently to stimulation with TLR3 ligands, whereas the responses from the fibroblasts were compromised. CONCLUSIONS: We report a novel TLR3 variant associated with recurrent zoster ophthalmicus. Toll-like receptor 3 responses that were unaffected in APCs but diminished in fibroblasts are in line with previous reports linking TLR3 deficiency with herpes simplex virus encephalitis. Mechanisms involving compromised viral sensing in infected cells may thus be central to the described immunodeficiency.

Interferon signature in immunosuppressed patients with lower respiratory tract infections: dosage on bronchoalveolar lavage.

BACKGROUND: Interferon signature (IS) is the measure of transcripts belonging to pathways of interferon activation. Viral infections can interfere with the interferon pathway, in particular herpesvirus present in immunocompromised hosts. The aim of our study was to evaluate if herpesvirus infections in immunocompromised patients with lower respiratory tract infections (LRTI) could lead to IS alterations. METHODS: We measured IS transcription of six genes on bronchoalveolar lavage of immunocompromised patients with LRTI (IFI27, IFI44, IFIT1, ISG15, RSAD2, SIGLEC1). Patients were divided in three groups based on Epstein-Barr virus (EBV) and other herpesviruses coinfections. RESULTS: We included 56 patients, 10 without and 17 with only EBV reactivation (respectively N and E groups) and 29 with EBV and other herpesviruses (group C). IS was higher in group C (P=0.01) compared to other ones, but single gene expressions were different among groups: IFI27 was higher whereas IFIT1 and ISG15 were lower in group C (P<0.05). CONCLUSIONS: The continuous stimulation of interferon cascade by herpesviruses enhances IS. The analysis of IS in immunocompromised population is possible by limiting the use of IFI27, IFIT1, ISG15 genes. Our preliminary results seem to indicate that IS is a useful biomarker of cellular response to herpesvirus infection in immunocompromised patients.

Impact of flavin-containing monooxygenase 3 and CYP2C19 genotypes on plasma disposition and adverse effects of voriconazole administered orally in immunocompromised patients.

Flavin-containing monooxygenase (FMO) 3 together with cytochrome P450 (CYP) 2C19 play a significant role in voriconazole N-oxidation. This study aimed to evaluate the influence of FMO3 and CYP2C19 genotypes on the plasma disposition and adverse effects of voriconazole in immunocompromised patients. Sixty-five Japanese immunocompromised patients receiving oral voriconazole were enrolled. Predose plasma concentrations of voriconazole and N-oxide were determined at day 5 or later. The adverse effects of voriconazole and the FMO3 and CYP2C19 genotypes were investigated. The patients with FMO3 E158K/E308G had a lower plasma concentration of voriconazole. The metabolic ratio to N-oxide was significantly higher in the FMO3 E158K/E308G group than in the wild group. In contrast, FMO3 V257M was not associated with the plasma concentration of voriconazole. No significant difference was observed in the saturation index, defined as a correlation coefficient of the regression line between the absolute plasma concentration of voriconazole and the inverse value of the metabolic ratio to N-oxide, between the FMO3 genotypes. CYP2C19 phenotype did not affect the plasma concentration and metabolic ratio of voriconazole. The saturation index of voriconazole rose in the order of CYP2C19 extensive, intermediate, and then poor metabolizer groups. However, the FMO3 and CYP2C19 genotypes and their associated voriconazole pharmacokinetics did not have an effect on the incidence of adverse effects. In conclusion, FMO3 E158K/E308G decreased the plasma concentration of voriconazole through its higher metabolic activity. The FMO3 genotype altered the plasma exposure of voriconazole, while the CYP2C19 phenotype affected the metabolic capacity in immunocompromised patients.

Frequency of the CYP2C19*17 polymorphism in a Chilean population and its effect on voriconazole plasma concentration in immunocompromised children.

Invasive fungal infections (IFIs) are the most frequent cause of morbidity and mortality in immunocompromised children. Voriconazole is the first-line antifungal choice in the treatment of IFIs like aspergillosis. Voriconazole pharmacokinetics vary widely among patients and voriconazole is metabolized mainly in the liver by the CYP2C19 enzyme, which is highly polymorphic. The CYP2C19*17 allele is characterized by the presence of four single nucleotide polymorphisms expressing an ultra-rapid enzyme phenotype with an accelerated voriconazole metabolism, is associated with low (sub-therapeutic) plasma levels in patients treated with the standard dose. Considering that in our center a high percentage of children have sub-therapeutic levels of voriconazole when treated with standard doses, we sought to determine the frequency of the CYP2C19*17 polymorphism (rs12248560) in a Chilean population and determine the association between voriconazole concentrations and the rs12248560 variant in immunocompromised children. First, we evaluated the frequency of the rs12248560 variant in a group of 232 healthy Chilean children, and we found that 180 children (77.6%) were non-carriers of the rs12248560 variant, 49 children (21.1%) were heterozygous carriers for rs12248560 variant and only 3 children (1.3%) were homozygous carriers for rs12248560 variant, obtaining an allelic frequency of 12% for variant in a Chilean population. To determine the association between voriconazole concentrations and the rs12248560 variant, we analyzed voriconazole plasma concentrations in a second group of 33 children treated with voriconazole. In these patients, carriers of the rs12248560 variant presented significantly lower voriconazole plasma concentrations than non-carriers (p = 0,011). In this study, we show the presence of the rs12248560 variant in a Chilean population and its accelerating effect on the pharmacokinetics of voriconazole in pediatric patients. From these data, it would be advisable to consider the variant of the patient prior to calculating the dosage of voriconazole.

Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination.

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.

Immune Dysfunction and Risk of Infection in Chronic Kidney Disease.

Cardiovascular disease and infections are directly or indirectly associated with an altered immune response, which leads to a high incidence of morbidity and mortality, and together, they account for up to 70% of all deaths among patients with chronic kidney dysfunction. Impairment of the normal reaction of the innate and adaptive immune systems in chronic kidney disease predisposes patients to an increased risk of infections, virus-associated cancers, and a diminished vaccine response. On the other hand, an abnormal, exaggerated reaction of the immune systems can also occur in this group of patients, resulting in increased production and decreased clearance of proinflammatory cytokines, which can lead to inflammation and its sequelae (eg, atherosclerotic cardiovascular disease). Epigenetically, modifications in hematopoietic stem cells involving a shift from lymphoid to myeloid cell lineage may underlie uremia-associated immunological senescence, which is not reversed by renal replacement therapy, including kidney transplantation. Measures aimed at attenuating the immune abnormalities in chronic kidney disease/end-stage renal disease should be an area of focused research as this could potentially lead to a better understanding and, thus, development of therapies that could reduce the disastrously high death rate in this patient population. The aim of the present article is to review the characteristics, causes, and mechanisms of the immune dysfunction related to chronic kidney disease.