Late-onset enteric virus infection associated with hepatitis (EVAH) in transplanted SCID patients.

BACKGROUND: Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE: We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS: We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS: Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS: Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Small bowel disease prevalence on video capsule endoscopy in chronic granulomatous disease-associated inflammatory bowel disease.

BACKGROUND AND AIM: Patients with chronic granulomatous disease (CGD) may develop inflammatory bowel disease (IBD). Characterization of small bowel disease in this cohort is scarce. Here, we sought to determine the prevalence and characteristics of small bowel disease and evaluate the clinical utility of video capsule endoscopy (VCE) for its diagnosis. METHODS: A retrospective study was performed on patients with CGD who were evaluated for gastrointestinal disease with VCE as a part of ongoing natural history studies at a single academic center. VCEs were reviewed for inflammatory findings and severity of disease utilizing the Capsule Endoscopy Crohn's Disease Activity Index. Radiographic studies and endoscopies performed within 30 days of VCE were compared with small bowel inflammatory findings. RESULTS: Twenty-six VCEs corresponding to 25 patients were found. The majority of patients were male and White; mean age was 28 years old. The majority (85%) demonstrated presence of small bowel inflammatory findings on VCE including strictures, ulcers, erosions, and erythema. Duodenal and ileal inflammatory disease on endoscopy did not correlate with disease on VCE. Moderate-severe colonic disease correlated with moderate-severe disease on VCE. Radiography did not correlate with disease on VCE. Prolonged small bowel transit time correlated with moderate-severe small bowel disease. CONCLUSIONS: Small bowel IBD was highly prevalent in this cohort of patients with CGD. Limitations included small sample size. Given that radiology and duodenal/ileal disease did not correlate with VCE findings, VCE-driven investigation of small bowel disease should be considered in patients with CGD-associated IBD, particularly those with colonic disease.

Poor T-cell receptor ß repertoire diversity early posttransplant for severe combined immunodeficiency predicts failure of immune reconstitution.

BACKGROUND: Development of a diverse T-cell receptor ß (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution. OBJECTIVES: We investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID. METHODS: We used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements. RESULTS: TRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention. CONCLUSIONS: TRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Safety and Efficacy of Ustekinumab in the Inflammatory Bowel Disease of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by mutations encoding the NADPH oxidase complex.1 Those affected are at increased risk of bacterial and fungal infections and require antimicrobial prophylaxis. Dysregulated inflammation may cause inflammatory bowel disease (IBD), termed CGD-associated IBD or CGD colitis, a distinct entity from Crohn's disease (CD) or ulcerative colitis (UC).

Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair.

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

MAGT1 messenger RNA-corrected autologous T and natural killer cells for potential cell therapy in X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia disease.

BACKGROUND AIM: X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect' (XMEN) disease is caused by mutations in the magnesium transporter 1 (MAGT1) gene. Loss of MAGT1 function results in a glycosylation defect that abrogates expression of key immune proteins such as the NKG2D receptor on CD8+ T and NK cells, which is critical for the recognition and killing of virus-infected and transformed cells, a biomarker for MAGT1 function. Patients with XMEN disease frequently have increased susceptibility to EBV infections and EBV-associated B cell malignancies, for which no specific treatment options are currently available. Experimental transfer of donor EBV-specific cytotoxic T cells may be beneficial but carries the risks of eliciting alloimmune responses. An approach for cell therapy to address viral infections and associated complications that avoids the risks of alloimmunity is needed. METHODS: Here the authors assess the feasibility and efficiency of correcting autologous lymphocytes from XMEN patients by MAGT1 mRNA electroporation (EP) that avoids genomic integration and can be scaled for clinical application. RESULTS AND CONCLUSIONS: Restoration of NKG2D expression was demonstrated in XMEN patient lymphocytes after MAGT1 mRNA electroporation that reach healthy donor levels in CD8+ T and NK cells at 1-2 days after EP. NKG2D expression persisted at ∼50% for 2 weeks after EP. Functionally, mRNA-correction of XMEN NK cells rescued cytotoxic activity also to healthy donor NK cell level. The restored NKG2D receptor expression and function were unaffected by cryopreservation, which will make feasible repeat infusions of MAGT1 mRNA-corrected autologous XMEN CD8+ T and NK cells for potential short term therapy for XMEN patients without the risks of alloimmunization.

Progressive B Cell Loss in Revertant X-SCID.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction.

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Enhancers are major targets for murine leukemia virus vector integration.

UNLABELLED: Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34(+) hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE: We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors.

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

Mobilization characteristics and strategies to improve hematopoietic progenitor cell mobilization and collection in patients with chronic granulomatous disease and severe combined immunodeficiency.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF)-mobilized autologous hematopoietic progenitor cells (HPCs) may be collected by apheresis of patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency (SCID) for use in gene therapy trials. CD34+ cell mobilization has not been well characterized in such patients. STUDY DESIGN AND METHODS: We retrospectively evaluated CD34+ cell mobilization and collection in 73 consecutive CGD and SCID patients and in 99 age-, weight-, and G-CSF dose-matched healthy allogeneic controls. RESULTS: In subjects aged not more than 20 years, Day 5 preapheresis circulating CD34+ counts were significantly lower in CGD and SCID patients than in controls; mean peak CD34+ cell counts were 58 × 10(6) , 64 × 10(6) , and 87 × 10(6) /L, respectively (p = 0.01). The SCIDs had lower CD34+ collection efficiency than CGDs and controls; mean efficiencies were 40, 63, and 57%, respectively (p = 0.003). In subjects aged more than 20 years, the CGDs had significantly lower CD34+ cell mobilization than controls; mean peak CD34+ cell counts were 41 × 10(6) and 113 × 10(6) /L, respectively (p < 0.0001). In a multivariate analysis, lower erythrocyte sedimentation rate (ESR) at mobilization was significantly correlated with better CD34+ cell mobilization (p = 0.007). In SCIDs, CD34 collection efficiency was positively correlated with higher red blood cell (RBC) indices (mean RBC volume, R(2) = 0.77; mean corpuscular hemoglobin [Hb], R(2) = 0.94; mean corpuscular Hb concentration, R(2) = 0.7; p < 0.007) but not Hb. CONCLUSIONS: CGD and SCID populations are characterized by significantly less robust CD34+ HPC mobilization than healthy controls. The presence of active inflammation or infection as suggested by an elevated ESR may negatively impact mobilization. Among SCIDs, markedly reduced CD34 collection efficiencies were related to iron deficiency, wherein decreased RBC size and density may impair apheresis cell separation mechanics.

CXCR4/IgG-expressing plasma cells are associated with human gastrointestinal tissue inflammation.

BACKGROUND: We previously reported abnormalities in circulating B cells in patients with chronic granulomatous disease (CGD) and those with HIV infection. Gastrointestinal complications are common to both diseases and likely involve perturbation of immune cells, including plasma cells (PCs). IgA is the most abundant immunoglobulin in the human body, with roles in protection and maintenance of intestinal homeostasis. IgA is produced primarily by PCs residing in mucosal tissues that are also thought to circulate in the blood. OBJECTIVE: We sought to characterize and compare PCs in patients with infectious (HIV) and noninfectious (CGD and Crohn disease) diseases that have been associated with intestinal inflammation. METHODS: Phenotypic and transcriptional analyses were performed on cells isolated from the blood and colon. RESULTS: IgA-secreting CCR10-expressing PCs predominated in the guts of healthy subjects, whereas in patients with HIV, CGD, and Crohn disease, there was a significant increase in the proportion of IgG-secreting PCs. Where intestinal inflammation was present, IgG-secreting PCs expressed reduced levels of CCR10 and increased levels of CXCR4. The intensity of CXCR4 expression correlated with the frequency of IgG-expressing PCs and the frequency of CXCR4(+)/IgG(+) PCs was associated with the severity of intestinal inflammatory disease yet distinct from PCs and plasmablasts circulating in the blood. CONCLUSIONS: These findings suggest that regardless of the underlying disease, the presence of CXCR4(+)/IgG(+) PCs in the gut is a strong yet localized indicator of intestinal inflammation. Furthermore, our findings suggest that CXCR4(+)/IgG(+) PCs might play a role in immune cell homeostasis during inflammatory processes of the gut.

Paravertebral mushroom: identification of a novel species of Phellinus as a human pathogen in chronic granulomatous disease.

We describe a case of paravertebral abscess caused by a Phellinus sp. in a boy with chronic granulomatous disease. Sequence-based identification of this mold, a new agent of disease, suggests a close relation to Phellinus umbrinellus.