Elizabeth M. Staley

Role of postnatal acquisition of the intestinal microbiome in the early development of immune function.

Objectives: Therapy with broad-spectrum antibiotics is a common practice for premature infants. This treatment can reduce the biodiversity of the fecal microbiota and may be a factor in the cause of necrotizing enterocolitis. In contrast, probiotic treatment of premature infants reduces the incidence of necrotizing enterocolitis. We hypothesized that 1 mechanism for these observations is the influence of bacteria on postnatal development of the mucosal immune system. Materials and Methods: Expression of immune molecules and microbial sensors was investigated in the postnatal mouse gastrointestinal tract by real-time polymerase chain reaction. Subsequently, 2-week-old specific pathogen-free and microbial-reduced (MR; antibiotic treated) mice were compared for immune molecule and microbial sensor expression, mesenteric lymph node T-cell numbers and activation, intestinal barrier function/permeability, systemic lymphocyte numbers, and T-cell phenotype commitment. Results: Toll-like receptor 2, 4, and 5 expression was highest in 2-week-old specific pathogen-free mice, and this expression was decreased in MR mice. There was no difference in intestinal tight-junctional function, as evaluated by fluorescein isothiocyanate-dextran uptake, but MR mice had increased bacterial translocation across the intestinal epithelial barrier. MR mice had significantly fewer splenic B cells and mesenteric lymph node CD4+ T cells, but there were normal numbers of splenic T cells. These systemic T cells from MR mice produced more interleukin-4 and less interferon-γ and IL-17, indicative of maintenance of the fetal, T-helper cell type 2 phenotype. Conclusions: The present study shows that intestinal commensal microbiota have an influence on early postnatal immune development. Determining specific bacteria and/or bacterial ligands critical for this development could provide insight into the mechanisms by which broad-spectrum antibiotics and/or probiotic therapy influence the development of the mucosal immune system and mucosal-related diseases.

Differential Susceptibility of P-glycoprotein Deficient Mice to Colitis Induction by Environmental Insults

Background: P‐glycoprotein (P‐gp), the product of the multidrug resistance gene (MDR), is an ATP‐dependent transmembrane pump, which is expressed in multiple cell lineages including epithelial and hematopoetic cells. The human MDR gene is located on chromosome 7 (7q21.1), a susceptibility loci for inflammatory bowel disease (IBD). A significant number of IBD patients carry mutations in this gene and P‐gp‐deficient FVB/N mice develop a severe spontaneous colitis, characterized by impaired intestinal barrier function and immune reactivity to intestinal bacterial antigens. Methods: In this work we explored the role of mouse strain, as well as environmental insults, on the development of colonic inflammation in the absence of P‐gp. Among the induction methods utilized, dextran sodium sulfate (DSS) disrupts the intestinal epithelium, while piroxicam is a nonsteroidal antiinflammatory (NSAID) drug that inhibits prostaglandin production and initiates colitis in IL10‐deficient animals. Helicobacter bilis is a known mediator of bacterial‐induced colitis. Results: We demonstrate that crossing this mutation onto the C57BL/6 strain confers protection from spontaneous colitis. C57BL/6. mdr1a‐deficient animals demonstrated increased histological inflammation, colonic shortening, fecal blood, and reduced body weight after 7 days of treatment with 2.25% DSS. C57BL/6. mdr1a‐deficient mice treated with piroxicam or infected with H. bilis showed no weight loss, or alterations in colonic histology. Conclusions: These data indicate that the effects of P‐gp deficiency are significantly modulated by background strain influences, but that the epithelium continues to have increased susceptibility to chemical injury in the C57BL/6 model.

Altered generation of induced regulatory T cells in the FVB.mdr1a-/- mouse model of colitis.

The FVB.mdr1a−/− mouse, lacking the small molecule pump P-glycoprotein (P-gp), is a commonly used model for the study of spontaneous T cell–mediated colitis. In addition, MDR1 polymorphisms and P-gp deficiency in humans have been linked to the development of ulcerative colitis. We now demonstrate that mice with P-gp deficiency have decreased levels of Foxp3+ regulatory T cells (Tregs) in the intestinal lamina propria. This decrease is not due to either increased Treg apoptosis, altered Treg trafficking, or enhanced Treg plasticity to become Foxp3+IL-17+ cells. Instead, P-gp deficiency appears to restrict the development of induced Treg cells (iTregs), as fewer Foxp3+ iTregs developed from naive FVB.mdr1a−/− T cells both upon transforming growth factor-β (TGF-β) treatment in vitro and after adoptive transfer into FVB.rag2−/− recipients. Rather, in vitro TGF-β treatment results in a IL-17+CD4+ T cell. This failure of iTregs to develop explains the decrease in Foxp3+ Tregs in the FVB.mdr1a−/− intestine, representing a need to investigate this novel disease mechanism in human inflammatory bowel disease patients with MDR1 polymorphisms.

Critical role for P-glycoprotein expression in hematopoietic cells in the FVB.Mdr1a(-/-) model of colitis.

Objective: P-glycoprotein (P-gp), the functional product of the multidrug resistance gene (MDR), is a transmembrane protein that extrudes substrates from the intracellular environment. P-gp is expressed on the apical surface of epithelial cells and on cells from the hematopoietic lineage. Human MDR polymorphisms have been associated with the increased risk of inflammatory bowel disease, and FVB/N animals deficient in mdr1a expression develop spontaneous colitis. Previous studies using adult bone marrow chimeras indicated that colitis development in this animal model was contingent on P-gp deficiency in radiation-resistant epithelial cells; however, the use of adult animals may mask the role of hematopoietic immune cells in colitis initiation, due to preexisting epithelial abnormalities. Subjects and Methods: To assess the importance of P-gp expression in intestinal epithelial and hematopoietic-derived cells on colitis induction in FVB.mdr1a−/− animals, we developed a neonatal model of bone marrow reconstitution. FVB/N and FVB.mdr1a−/− adult and neonatal animals were lethally irradiated and reconstituted with bone marrow from FVB/N or FVB.mdr1a−/− donors. Animals were observed for 20 weeks. Results: Adult FVB/N animals deficient in P-gp expression in hematopoietically derived immune cells developed colitis similar to adult animals deficient in P-gp expression in radiation-resistant epithelial/stromal cells. Neonatal animals deficient in P-gp expression in hematopoietically derived immune cells developed a more histologically significant colitis than those deficient in P-gp expression in epithelial tissue. Conclusions: The use of a neonatal model of bone marrow reconstitution has revealed a critical role for P-gp expression in hematopoietically derived immune cells in colitis development in the FVB.mdr1a−/− model.

An update on ABO incompatible hematopoietic progenitor cell transplantation

Hematopoietic progenitor cell (HPC) transplantation has long been established as the optimal treatment for many hematologic malignancies. In the setting of allogenic HLA matched HPC transplantation, greater than 50% of unrelated donors and 30% of related donors demonstrate some degree of ABO incompatibility (ABOi), which is classified in one of three ways: major, minor, or bidirectional. Major ABOi refers to the presence of recipient isoagglutinins against the donors A and/or B antigen. Minor ABOi occurs when the HPC product contains the isoagglutinins targeting the recipients A and/or B antigen. Bidirectional refers to the presence of both major and minor ABOi. Major adverse events associated with ABOi HPC transplantation includes acute and delayed hemolysis, pure red cell aplasia, and delayed engraftment. ABOi HPC transplantation poses a unique challenge to the clinical transplantation unit, the HPC processing lab, and the transfusion medicine service. Therefore, it is essential that these services actively communicate with one another to ensure patient safety. This review will attempt to globally address the challenges related to ABOi HPC transplantation, with an increased focus on aspects related to the laboratory and transfusion medicine services.

Plasma Transfusion Demystified: A Review of the Key Factors Influencing the Response to Plasma Transfusion

Many studies have suggested that inappropriate plasma usage is common. An important factor contributing to futile plasma administration in most patients is the nonlinear relationship between coagulation-factor levels and the volume of plasma transfused. In this review, a validated mathematical model and data from the literature will be used to illuminate 3 key properties of plasma transfusion. Those properties are as follows: the effect of plasma transfusion on international normalized ratio (INR) is transient; for the same volume of transfused plasma, a greater reduction in INR is observed at higher initial INRs; and the effect of plasma transfusion on INR correction (ie, the difference between initial and final INRs) diminishes as more plasma is transfused. Frequent misunderstanding of these properties may contribute to inappropriate plasma usage. Therefore, this review will assist physicians in navigating these common pitfalls. Stronger understanding of these principles may result in a reduction of inappropriate plasma transfusions, thus potentially enhancing patient safety and reducing healthcare costs.

ADAMTS13 test and/or PLASMIC clinical score in management of acquired thrombotic thrombocytopenic purpura: a cost-effective analysis

The ADAMTS13 test distinguishes thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies (TMAs). The PLASMIC score helps determine the pretest probability of ADAMTS13 deficiency. Due to inherent limitations of both tests, and potential adverse effects and cost of unnecessary treatments, we performed a cost‐effectiveness analysis (CEA) investigating the benefits of incorporating an in‐hospital ADAMTS13 test and/or PLASMIC score into our clinical practice.

Murine P-glycoprotein Deficiency Alters Intestinal Injury Repair and Blunts Lipopolysaccharide-Induced Radioprotection

P-glycoprotein (P-gp) has been reported to increase stem cell proliferation and regulate apoptosis. Absence of P-gp results in decreased repair of intestinal epithelial cells after chemical injury. To further explore the mechanisms involved in the effects of P-gp on intestinal injury and repair, we used the well-characterized radiation injury model. In this model, injury repair is mediated by production of prostaglandins (PGE2) and lipopolysaccharide (LPS) has been shown to confer radioprotection. B6.mdr1a–/– mice and wild-type controls were subjected to 12 Gy total body X-ray irradiation and surviving crypts in the proximal jejunum and distal colon were evaluated 3.5 days after irradiation. B6.mdr1a–/– mice exhibited normal baseline stem cell proliferation and COX dependent crypt regeneration after irradiation. However, radiation induced apoptosis was increased and LPS-induced radioprotection was blunted in the C57BL6.mdr1a–/– distal colon, compared to B6 wild-type controls. The LPS treatment induced gene expression of the radioprotective cytokine IL-1α, in B6 wild-type controls but not in B6.mdr1a–/– animals. Lipopolysaccharid-induced radioprotection was absent in IL-1R1–/– animals, indicating a role for IL-1α in radioprotection, and demonstrating that P-gp deficiency interferes with IL-1α gene expression in response to systemic exposure to LPS.

Anti–Blood Group Antibodies in Intravenous Immunoglobulin May Complicate Interpretation of Antibody Titers in ABO-Incompatible Transplantation

Patients receiving ABO‐incompatible (ABOi) kidney transplants are treated before and after transplant with combination therapy, such as intravenous immunoglobulin (IVIG) and therapeutic plasma exchange, to prevent allograft rejection by reducing anti‐A and anti‐B titers. Although generally considered safe, it is well known that commercial IVIG products contain detectable anti‐A and anti‐B, which can be associated with hemolysis. Different preparative manufacturing techniques during the production of IVIG affect ABO antibody levels in IVIG preparations; therefore, some manufacturers now use new methods to reduce anti‐A/B levels at the preproduction stage. The variations in implementing these strategies creates the potential for significant variation in antibody titers between products and, in some cases, even between lots of the same IVIG product. We report a case of persistently elevated anti‐A titers in an ABOi kidney transplant recipient associated with elevated ABO antibody titers present in the preparation of IVIG used at our facility.

Therapeutic plasma exchange For Hashimoto's encephalopathy

To the Editor, Hashimoto’s encephalopathy (HE) is a rare neuropsychiatric disorder associated with increased anti-thyroid, and most frequently, anti-thyroperoxidase (TPO) antibodies. It is estimated to occur in 2.1/100 000 people, with fewer than 200 cases reported. The clinical presentation includes stroke-like findings, seizures, psychosis, coma, and potentially death, typically without concurrent thyroid disease. Although antithyroid antibodies are considered a biomarker of the disorder, their role in the underlying disease pathogenesis remains uncertain, and the antibody titers may not correlate with disease severity.2–4 Previously reported successful treatments include corticosteroids, immunosuppressants, and therapeutic plasma exchange (TPE). Based solely on case reports and a case series of 15 patients, the 2016 American Society for Apheresis (ASFA) Guidelines listed TPE, for the first time, as