Geraldine G. Miller

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

Differential Expression of the IFN-γ-Inducible CXCR3-Binding Chemokines, IFN-Inducible Protein 10, Monokine Induced by IFN, and IFN-Inducible T Cell α Chemoattractant in Human Cardiac Allografts: Association with Cardiac Allograft Vasculopathy and Acute Rejection

CXCR3 chemokines exert potent biological effects on both immune and vascular cells. The dual targets suggest their important roles in cardiac allograft vasculopathy (CAV) and rejection. Therefore, we investigated expression of IFN-inducible protein 10 (IP-10), IFN-inducible T cell α chemoattractant (I-TAC), monokine induced by IFN (Mig), and their receptor CXCR3 in consecutive endomyocardial biopsies (n = 133) from human cardiac allografts and corresponding normal donor hearts (n = 11) before transplantation. Allografts, but not normal hearts, contained IP-10, Mig, and I-TAC mRNA. Persistent elevation of IP-10 and I-TAC was associated with CAV. Allografts with CAV had an IP-10-GAPDH ratio 3.7 ± 0.8 compared with 0.8 ± 0.2 in those without CAV (p = 0.004). Similarly, I-TAC mRNA levels were persistently elevated in allografts with CAV (6.7 ± 1.9 in allografts with vs 1.5 ± 0.3 in those without CAV, p = 0.01). In contrast, Mig mRNA was induced only during rejection (2.4 ± 0.9 with vs 0.6 ± 0.2 without rejection, p = 0.015). In addition, IP-10 mRNA increased above baseline during rejection (4.1 ± 2.3 in rejecting vs 1.8 ± 1.2 in nonrejecting biopsies, p = 0.038). I-TAC did not defer significantly with rejection. CXCR3 mRNA persistently elevated after cardiac transplantation. Double immunohistochemistry revealed differential cellular distribution of CXCR3 chemokines. Intragraft vascular cells expressed high levels of IP-10 and I-TAC, while Mig localized predominantly in infiltrating macrophages. CXCR3 was localized in vascular and infiltrating cells. CXCR3 chemokines are induced in cardiac allografts and differentially associated with CAV and rejection. Differential cellular distribution of these chemokines in allografts indicates their central roles in multiple pathways involving CAV and rejection. This chemokine pathway may serve as a monitor and target for novel therapies to prevent CAV and rejection.

Interlaboratory Comparison of Cytomegalovirus Viral Load Assays

To assess interlaboratory variability in qualitative and quantitative cytomegalovirus (CMV) viral load (VL) testing, we distributed a panel of samples to 33 laboratories in the USA, Canada and Europe who performed testing using commercial reagents (n = 17) or laboratory‐developed assays (n = 18). The panel included two negatives, seven samples constructed from purified CMV nucleocapsids in plasma (2.0–6.0 log10 copies/mL) and three clinical plasma samples. Interlaboratory variation was observed in both actual (range, 2.0–4.0 log10 copies/mL) and self‐reported lower limits of detection (range, 1.0–4.0 log10 copies/mL). Variation observed in reported results for individual samples ranged from 2.0 log10 (minimum) to 4.3 log10 (maximum). Variation was greatest at low VLs. Assuming ± 0.5 log10 relative to the expected result represents an acceptable result, 57.6% of results fell within this range. Use of commercially available reagents and procedures was associated with less variability compared with laboratory‐developed assays. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). The significant interlaboratory variability in CMV VL observed may be impacting patient care and limiting interinstitutional comparisons. The creation of an international reference standard for CMV VL assay calibration would be an important step in quality improvement of this laboratory tool.

In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1.

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C

Streptococcus pyogenes (group A Streptococcus) has re-emerged in recent years as a cause of severe human disease. Because extracellular products are involved in streptococcal pathogenesis, we explored the possibility that a disease isolate expresses an uncharacterized superantigen. We screened culture supernatants for superantigen activity with a major histocompatibility complex class II-dependent T cell proliferation assay. Initial fractionation with red dye A chromatography indicated production of a class II-dependent T cell mitogen by a toxic shock-like syndrome (TSLS) strain. The amino terminus of the purified streptococcal superantigen was more homologous to the amino termini of staphylococcal enterotoxins B, C1, and C3 (SEB, SEC1, and SEC3), than to those of pyrogenic exotoxins A, B, C or other streptococcal toxins. The molecule, designated SSA, had the same pattern of class II isotype usage as SEB in T cell proliferation assays. However, it differed in its pattern of human T cell activation, as measured by quantitative polymerase chain reaction with V beta-specific primers. SSA activated human T cells that express V beta 1, 3, 15 with a minor increase of V beta 5.2-bearing cells, whereas SEB activated V beta 3, 12, 15, and 17-bearing T cells. Immunoblot analysis of 75 disease isolates from several localities detected SSA production only in group A streptococci, and found that SSA is apparently confined to only three clonal lineages as defined by multilocus enzyme electrophoresis typing. Isolates of one of these lineages, (electrophoretic type 2) are strongly associated with TSLS. The data identify SSA as a novel streptococcal superantigen that appears to be more related structurally to staphylococcal enterotoxins than to streptococcal exotoxins. Because abundant SSA production is apparently confined to only three streptococcal clonal lineages, the data also suggest that the SSA gene has only recently been acquired by S. pyogenes.

Drug Therapy in the Heart Transplant Recipient Part II: Immunosuppressive Drugs

Received March 16, 2004; revision received July 23, 2004; accepted September 30, 2004. Part I of this series describes the mechanisms and types of rejection and the intravenous immunosuppressive drugs commonly used for induction or antirejection therapy. In this article, we review the commonly used oral immunosuppressive drugs. Intravenous corticosteroid methylprednisolone is included in the discussion of corticosteroids. Table 1 gives trade names, pharmacology, necessary adjustments for renal or hepatic dysfunction, and dosing and general monitoring guidelines for drugs described in this section. Table 2 lists the major adverse effects of immunosuppressive drugs described in Parts I and II of this review and provides an estimate of their relative frequency. View this table: TABLE 1. Commonly Used Oral (and Intravenous) Immunosuppressive Drugs View this table: TABLE 2. Major Adverse Effects of Immunosuppressive Drugs Steroids, among the first immunosuppressive agents used in clinical transplantation, have remained an important component of induction, maintenance, and rejection regimens. ### Mechanism of Action Glucocorticoids are potent immunosuppressive and antiinflammatory agents (the Figure). They diffuse freely across cell membranes and bind to high-affinity cytoplasmic glucocorticoid receptors. The glucocorticoid receptor–steroid complex translocates to the nucleus, where it binds to a glucocorticoid response element within the DNA.1 The glucocorticoid receptor–steroid complex may also bind to other regulatory elements, inhibiting their binding to DNA. Both actions cause transcriptional regulation, thereby altering the expression of genes involved in immune and inflammatory response. Glucocorticoids affect the number, distribution, and function of all types of leukocytes (T and B lymphocytes, granulocytes, macrophages, and monocytes), as well as endothelial cells.2 The major effect on lymphocytes appears to be mediated by inhibition of 2 transcription factors, activator protein-1 and nuclear factor (NF) κ-B.3,4 This affects the expression of a number of genes, including those for growth factors, cytokines, CD40 ligand, GM-CSF, and adhesion and myosin heavy chain molecules.2 In nonlymphoid …

Drug therapy in the heart transplant recipient: part I: cardiac rejection and immunosuppressive drugs.

Survival after heart transplantation has improved considerably over the past 20 years. Half of all patients now live >9 years, and ≈25% live ≥17 years.1 Currently, ≈20 000 heart transplant recipients live in the United States.2 Improved longevity means prolonged immunosuppression and the concomitant use of drugs to prevent or treat the long-term complications of immunosuppressive agents, such as infection, obesity, hypertension, hyperlipidemia, renal insufficiency, diabetes, osteoporosis, gout, and malignancies. In 1989, heart transplant recipients surviving 1 year were reported to be taking 16±6 drug doses per day (prescription and nonprescription).3 In 2001, heart transplant recipients surviving an average of 76 months were taking 7 prescription drugs (range, 2 to 14), along with a number of nonprescription drugs.4 Thus, despite prolonged survival, heart transplant recipients continue to take multiple medications. With the large number of heart transplant recipients in the community and the increasing number of immunosuppressive and nonimmunosuppressive drugs used by these patients, it is important that the general cardiologist understand these drugs, their side effects, and the very real potential for drug–drug interactions. These interactions may result in adverse events caused by supratherapeutic and subtherapeutic drug concentrations. In this series, we review mechanisms and types of rejection, immunosuppressive drugs commonly used in the heart transplant recipient, common medical problems after transplantation, and clinically significant drug–drug interactions. A brief review of known immunologic mechanisms leading to graft rejection highlights the action of individual immunosuppressive drugs, as well as the rationale for combination therapy5–8 (Figure). The rejection of a transplanted organ is primarily a T-lymphocyte (T-cell)–mediated event, although humoral (B-cell) responses also contribute. The exception is hyperacute rejection, which occurs when preformed antibodies to human leukocyte antigens (HLA) result in an immediate and catastrophic rejection. Immune recognition of donor antigens that differ from those of …

Interlaboratory comparison of epstein-barr virus viral load assays.

To assess interlaboratory variability in qualitative and quantitative Epstein‐Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory‐developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt‐3 cell lines diluted in plasma (1.30–5.30 log10 copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30–4.30 log10 copies/mL) and self‐reported (range, 1.70–3.30 log10 copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log10 (minimum) to 4.14 log10 (maximum). Variation was independent of dynamic range and use of commercial versus laboratory‐developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result ± 0.50 log10. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

Influenza Vaccination in the Organ Transplant Recipient: Review and Summary Recommendations

Influenza virus causes a spectrum of illness in transplant recipients with a high rate of lower respiratory disease. Seasonal influenza vaccination is an important public health measure recommended for transplant recipients and their close contacts. Vaccine has been shown to be safe and generally well tolerated in both adult and pediatric transplant recipients. However, responses to vaccine are variable and are dependent on various factors including time from transplantation and specific immunosuppressive medication. Seasonal influenza vaccine has demonstrated safety and no conclusive evidence exists for a link between vaccination and allograft dysfunction. Annually updated trivalent inactivated influenza vaccines have been available and routinely used for several decades, although newer influenza vaccination formulations including high‐dose vaccine, adjuvanted vaccine, quadrivalent inactivated vaccine and vaccine by intradermal delivery system are now available or will be available in the near future. Safety and immunogenicity data of these new formulations in transplant recipients requires investigation. In this document, we review the current state of knowledge on influenza vaccines in transplant recipients and make recommendations on the use of vaccine in both adult and pediatric organ transplant recipients.

Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori

Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strain‐specific restriction‐modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site‐specific methylation using cell‐free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell‐free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock‐treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.