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John D Roback

Publications and source records attributed to John D Roback.

At least 19 recordsLinked to original sources
PubMed2007

Transfusion-transmitted cytomegalovirus: lessons from a murine model.

Transfusion-transmitted cytomegalovirus (CMV) infection (TT-CMV) continues to complicate blood transfusion therapy, which can lead to severe morbidity or mortality in immunocompromised or immuno-immature recipients. The biological mechanisms that underlie TT-CMV (eg, viral latency in donor monocytes or stimulatory signals in the transfusion recipient leading to cytomegalovirus reactivation) are difficult to study in humans, but can be addressed in animal models. In this review, we discuss a mouse blood transfusion model, which can be used to investigate these issues as well as to validate methods to prevent TT-CMV in at-risk patients.

Animals↗
PubMed2006

The role of photochemical treatment with amotosalen and UV-A light in the prevention of transfusion-transmitted cytomegalovirus infections.

Primary cytomegalovirus (CMV) infection is usually asymptomatic in immunocompetent patients but can cause serious life-threatening complications in immunocompromised CMV-seronegative patients, including patients receiving a bone marrow or peripheral blood stem cell transplant, recipients of some solid-organ transplants, and low-birth-weight neonates. Current recommendations for preventing transfusion-transmitted CMV (TT-CMV) infection in these patients include exclusive use of CMV-seronegative and/or leukoreduced cellular blood components (red blood cells and platelets) for transfusion. However, breakthrough cases of TT-CMV still occur. Despite improving the safety of blood components, testing remains a reactive approach to blood safety. In contrast, pathogen inactivation technologies offer a proactive approach with the potential to further improve blood safety. To reduce the risks associated with platelet transfusions, a photochemical treatment (PCT) process using a combination of the psoralen amotosalen HCl and long-wavelength UV light has been developed and introduced into clinical practice in Europe. PCT has been shown to result in greater than 5.9-log reductions in infectivity of human CMV in platelet concentrates and to prevent the transfusion transmission of murine CMV in a mouse transfusion model. Thus, PCT pathogen inactivation may play a role in further reducing the incidence of TT-CMV infection in patients who are at risk for serious CMV disease. Because PCT is a technology that targets nucleic acids, it also offers a proactive process for the inactivation of a broad range of viral, bacterial, and protozoan pathogens in addition to CMV.

Animals↗
PubMed2006

Transfusion-transmitted cytomegalovirus (CMV) infections in a murine model: characterization of CMV-infected donor mice.

BACKGROUND: Donor and recipient mechanisms that modulate the incidence and severity of transfusion-transmitted cytomegalovirus (TT-CMV) are unclear. The kinetics of murine CMV (MCMV) infection in the peripheral blood of donor mice were investigated to determine the utility of this model for studying TT-CMV. STUDY DESIGN AND METHODS: BALB/cByJ mice, experimentally infected with Smith strain MCMV, were killed at serial time points up to 28 days after infection. Peritoneal exudate cells (PECs), peripheral blood white blood cells (WBCs), plasma, and marrow were tested for MCMV DNA with quantitative polymerase chain reaction (PCR), replication-competent virus with quantitative culture, and transcription of viral genes with reverse transcription (RT)-PCR targeted at the immediate-early 1 (ie1) gene. RESULTS: PECs, macrophages infected by MCMV shortly after intraperitoneal inoculation, demonstrated high mean levels of MCMV DNA (10(5)-10(7) genome equivalents [geqs]/10(5) PECs), virus production (10(1)-10(4) infectious virions/10(5) PECs), and ie1 gene transcription, demonstrating productive infection. In contrast, while MCMV loads averaged 10(4) to 10(6) geqs per 10(5) peripheral WBCs, all WBC samples were uniformly negative for MCMV ie1 expression by RT-PCR and for culturable virus, consistent with latent MCMV infection. Plasma and marrow showed lower viral loads than WBCs and PECs and were all negative by culture and RT-PCR analysis. CONCLUSIONS: Following experimental MCMV infection, murine peripheral blood WBCs appear to be latently infected with virus (MCMV DNA-positive; MCMV RNA-negative; MCMV culture-negative), similar to the latently infected human monocytes in peripheral blood of CMV-seropositive donors. These donor kinetics suggest that the experimental MCMV system can be used to effectively model the mechanisms of TT-CMV infections in humans.

Animals↗
PubMed2006

Recipient inflammation affects the frequency and magnitude of immunization to transfused red blood cells.

BACKGROUND: Most alloantigens on transfused red blood cells (RBCs) are weakly immunogenic, with only a 2 to 6 percent overall immunization rate even in patients receiving multiple transfusions. Although recipient genetics may contribute to responder and/or nonresponder status, in most cases HLA type does not predict humoral response to RBC antigens. In contrast, rates of alloimmunization do correspond to the underlying disease status of transfusion recipients, suggesting that acquired host factors may play an important role. In this context, it was hypothesized that the inflammatory status of a transfusion recipient would influence immunization to transfused RBCs. STUDY DESIGN AND METHODS: A novel murine model for alloimmunization to RBC antigens was developed with the mHEL mouse, which expresses hen egg lysozyme (HEL) as a model blood group antigen. Leukoreduced mHEL RBCs were transfused into wild-type recipient mice, and anti-HEL responses were monitored. To test the stated hypothesis, some recipient animals were injected with polyinosinic polycytidylic acid (poly(I:C)), a synthetic double-stranded RNA molecule that induces viral-like inflammation. RESULTS: Similar to the immunogenicity of most RBC antigens in humans, transfusion of mHEL RBCs into uninflamed mice was only a weak immunogen. In contrast, poly(I:C)-treated mice had a significant increase in both the frequency and the magnitude of alloimmunization to the mHEL antigen. CONCLUSIONS: These findings demonstrate that recipient inflammation with poly(I:C) significantly enhances humoral immunization to transfused alloantigens in a murine model. Moreover, these data suggest that the inflammatory status of human transfusion recipients may regulate the immunogenicity of transfused RBCs.

Animals↗
PubMed2006

Vaccine-enhanced donor lymphocyte infusion (veDLI).

Hematopoietic stem cell or bone marrow transplantation (HSCT/BMT) is curative in many cases of hemato-logical malignancy, but the post-transplant course is often complicated by delayed immune reconstitution that predisposes to opportunistic infections and disease recurrence. Furthermore, since HLA-matched donors cannot be found for almost half of all patients that would benefit from HSCT, donors mismatched at 2-3 HLA loci are increasingly being used, which is associated with elevated rates of opportunistic infections. Donor lymphocyte infusion (DLI) is a powerful and direct approach to improve post-transplant immune function. For example, DLI using enriched antiviral cytolytic effectors (CTLs) has been shown to reconstitute cellular immunity to cytomega-lovirus (CMV) and Epstein-Barr virus (EBV) and prevent viral disease following HSCT. However, because in vitro expansion and purification of CTLs is lengthy, labor-intensive, and costly, it is rarely used clinically to prevent and treat viral infections following HSCT. Active vaccination after allogeneic transplantation to stimulate in vivo expansion of donor and/or recipient CTLs has been proposed as an alternative method to rapidly reconstitute antiviral immunity, prevent viral disease, and reduce adverse sequelae of antiviral drugs. Fortunately, recent progress has been made in developing vaccines and methodologies that are both safe and effective when administered to immunocompromised HSCT recipients.

Graft Survival↗
PubMed2005

Nonhemolytic antibody-induced loss of erythrocyte surface antigen.

Transfusion of red blood cells (RBCs) into patients with anti-donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the FcgammaIII receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.

Animals↗
PubMed2005

Amotosalen-treated donor T cells have polyclonal antigen-specific long-term function without graft-versus-host disease after allogeneic bone marrow transplantation.

We have previously shown that amotosalen HCl (S-59 psoralen)-treated donor splenocytes, which have limited proliferative capacity in vitro, can protect major histocompatibility complex-mismatched bone marrow transplant (BMT) recipients from lethal murine cytomegalovirus infection without causing graft-versus-host disease. In this study, we further investigated the effects of amotosalen-treated donor T cells on immune reconstitution after allogeneic BMT. We were surprised to find that amotosalen-treated donor T cells persisted long-term in vivo, comprising 6% to 10% on average of the T-cell compartment of transplant recipients at 4 months after transplantation. Donor T cells derived from amotosalen-treated splenocytes were predominantly polyclonal CD44 hi/int CD8 + memory T cells and were functionally active, synthesizing interferon gamma in response to stimulation with murine cytomegalovirus antigen. Amotosalen-treated donor T cells, reisolated from BMT recipients' spleens >/=4 months after transplantation, proliferated in vitro, thus indicating repair of amotosalen-mediated DNA cross-links. Compared with infusion of untreated donor splenocytes, amotosalen-treated cells enhanced thymopoiesis by bone marrow-derived stem cells in BMT recipients. However, amotosalen treatment abrogated the thymopoietic activity of lymphoid progenitor cells among the donor splenocytes. Thus, infusion of amotosalen-treated donor T cells produced rapid immune reconstitution after major histocompatibility complex-mismatched BMT by transferring long-lived polyclonal memory T cells with antiviral activity and also by enhancing bone marrow-derived thymopoiesis. This is a novel approach to adoptive immunotherapy in allogeneic BMT.

Animals↗
PubMed2005

Comparison of cytomegalovirus polymerase chain reaction and serology for screening umbilical cord blood components.

BACKGROUND: Recipients of umbilical cord blood (UCB) transplants are susceptible to opportunistic infections, including cytomegalovirus (CMV). To prevent CMV transmission from UCB donors, most laboratories perform serology on corresponding maternal samples and quarantine units when the mother has immunoglobulin M (IgM) anti-CMV. STUDY DESIGN AND METHODS: UCB units and associated samples (UCB plasma and red cell pellet; maternal whole blood and serum) from two cord blood banks were tested with two validated CMV polymerase chain reaction assays (UL54 and UL93 targets). Results were compared with maternal CMV serology (IgG and IgM). RESULTS: Only 4 of 48 (8.3%) quarantined CMV IgM-positive units were also CMV nucleic acid testing (NAT)-positive (651-68,600 copies/mL). In contrast, 1 of 200 "CMV-safe" UCB units (CMV IgM-equivocal or -negative) had CMV DNA (0.5%). The corresponding maternal samples were CMV NAT-negative. Positive maternal IgM serology demonstrates only modest sensitivity (80%) and specificity (82%) and poor positive predictive value (8%), when correlated with the presence of CMV DNA in UCB units. CONCLUSION: CMV NAT may be a useful adjunct to serologic screening, potentially reducing wastage of IgM-positive and NAT-negative units while also detecting potentially infectious units that would pass serologic screening. A prospective clinical trial to further evaluate the role of CMV NAT in UCB transplantation appears warranted.

Cells, Cultured↗
PubMed2004

Effect of mediators of innate immunity and inflammation on CD8+ veto cells.

BACKGROUND: Methods of allotolerance induction that are successful in mice often fail in primates. Activators of innate immunity derived from microbial pathogens, which are present in primate populations but not in pathogen-free mouse colonies, have been shown to overcome tolerance induction by co-stimulatory blockade or regulatory T cells. Unlike other strategies, veto cells promote long-term allograft survival in primates. Accordingly, the authors hypothesized that veto cells are resistant to the effects of innate immune activation. METHODS: Using mixed lymphocyte reactions as a model of alloimmunization, the authors assessed the effect of inflammatory mediators on veto cell activity. RESULTS.: The authors demonstrate that the activity of veto cells is unaltered by lipopolysaccharide, double-stranded RNA, or interferon-gamma. Tumor necrosis factor-alpha modestly inhibited the veto effect, but only when veto cells were limiting. CONCLUSIONS: These findings may help to explain why veto cell-based therapies are more effective than other approaches in primate populations.

Animals↗
PubMed2004

Leukoreduction filtration of blood with sickle cell trait.

Leukoreduction of cellular blood products by filtration has been shown to decrease the incidence of febrile nonhemolytic transfusion reactions, transmission of leukocyte-associated viruses, and HLA alloimmunization. However, the increasing popularity of leukofiltering blood products over the past few years has highlighted specific filtration failures associated with sickle trait (hemoglobin AS) blood. Sickle trait blood does not filter adequately, which leads to prolonged or incomplete filtration, often with a higher number of postfiltration leukocytes in the unit than the mandated minimal residual volume of <1 to 5 x 10(6). This review of the literature highlights various parameters that affect the adequacy of filtration of blood products, including effects of temperature, pH, osmolarity, type of anticoagulant, time of storage, and oxygen saturation of the blood unit. A combination of these factors likely contributes to the frequent filtration failure of sickle trait products. Although blood units are not routinely screened for sickle cell hemoglobin, administration of units with sickle trait red blood cells can be disadvantageous to certain patient populations. This review concludes with a discussion of different approaches to screening blood units for sickle cell trait.

Anticoagulants↗
PubMed2004

Improved method for fluorescence cytometric immunohematology testing.

BACKGROUND: A method for accurate immunohematology testing by fluorescence cytometry (FC) was previously described. Nevertheless, the use of vacuum filtration to wash RBCs and a standard-flow cytometer for data acquisition hindered efforts to incorporate this method into an automated platform. STUDY DESIGN AND METHODS: A modified procedure was developed that used low-speed centrifugation of 96-well filter plates for RBC staining. Small-footprint benchtop capillary cytometers (PCA and PCA-96, Guava Technologies, Inc.) were used for data acquisition. Authentic clinical samples from hospitalized patients were tested for ABO group and the presence of D antigen (n = 749) as well as for the presence of RBC alloantibodies (n = 428). Challenging samples with mixed-field reactions and weak antibodies were included. Results were compared to those obtained by column agglutination technology (CAT), and discrepancies were resolved by standard tube methods. Detailed investigations of FC sensitivity and reproducibility were also performed. RESULTS: The modified FC method with the PCA determined the correct ABO group and D type for 98.7 percent of 520 samples, compared to 98.8 percent for CAT (p > 0.05). No-type-determined (NTD) rates were 1.2 percent for both methods. In testing for unexpected alloantibodies, FC determined the correct result for 98.6 percent of 215 samples, compared to 96.3 percent for CAT (p > 0.05). When samples were automatically acquired in the 96-well plate format with the PCA-96, 98.7 percent of 229 samples had correct ABO group and D type determined by FC, compared to 97.4 percent for CAT (p > 0.05). NTD rates were 0.9 and 2.6 percent, respectively. Antibody screens were accurate for 99.1 percent of 213 samples with the PCA-96, compared to 99.5 percent for CAT (p > 0.05). Further investigations demonstrated that FC with the PCA-96 was better than CAT at detecting weak anti-A (p < 0.0001) and alloantibodies. CONCLUSIONS: An improved method for FC immunohematology testing has been described. This assay was comparable in accuracy to standard CAT techniques, but had better sensitivity for detecting weak antibodies and was superior in detecting mixed-field reactions (p < 0.005). The FC method demonstrated excellent reproducibility. The compatibility of this assay with the PCA-96 capillary cytometer with plate-handling capabilities should simplify development of a completely automated platform.

Autoantibodies↗
PubMed2004

Photochemical treatment of platelet concentrates with amotosalen hydrochloride and ultraviolet A light inactivates free and latent cytomegalovirus in a murine transfusion model.

BACKGROUND: A photochemical treatment (PCT) process utilizing amotosalen hydrochloride and long wavelength UVA light has been developed to inactivate pathogens in PLTs. This study investigated the effects of amotosalen/UVA treatment on free and latent murine CMV (MCMV) in PLT preparations using a murine model of transfusion-transmitted CMV (TT-CMV). STUDY DESIGN AND METHODS: In a model of latent MCMV infection, "donor" mice received 1 x 10(6) plaque-forming units (PFUs) MCMV and were rested 14 days. Subsequently harvested, pooled, and washed WBCs were PCR positive for MCMV. Murine WBC doses of 1 x 10(4), 1 x 10(5), and 1 x 10(6) were added to human apheresis PLTs in 35 percent autologous plasma and 65 percent PLT AS (PAS). The WBC-PLT products were treated with 150 micro mol/L amotosalen and 0.6 J per cm2 UVA and transfused via tail vein injection into recipient mice. Recipients were killed on Day 14. Blood and spleens were collected and assayed for MCMV by PCR. In a parallel model of active infection with free virus, human PLT in 35 percent autologous plasma and 65 percent PAS were dosed with 1 x 10(5) and 1 x 10(6) PFUs of MCMV. All other procedures were as described above. RESULTS: In the absence of amotosalen/UVA-pretreatment, transfusion of PLT latently or actively infected with MCMV produced TT-CMV in a dose-dependent fashion. In contrast, all transfusion recipients of identical PLT preparations pretreated with amotosalen/UVA were uniformly PCR negative for MCMV (abrogation of TT-CMV; p < 0.05). CONCLUSIONS: PCT of PLT preparations with the specified doses of amotosalen hydrochloride and UVA light prevents transfusion transmission of free and latent MCMV in a murine model. These results suggest that PCT of human PLTs with amotosalen/UVA should also effectively abrogate TT-CMV in the clinical setting.

Animals↗
PubMed2004

Description and investigation of white particulate matter in additive solution-1 red blood cell units.

BACKGROUND: In January 2003, "white particulate matter" (WPM) was transiently observed in red blood cell (RBC) units collected predominately in the southeastern US. In this report, these events, their chronology, pertinent observations and investigations, and summaries and conclusions associated with WPM during the 2-week observation period are described. CHRONOLOGY AND INVESTIGATIONS: On January 27, 2003, WPM was first identified in RBCs; by January 31, 2003, 110 RBC units containing WPM had been identified. Elective surgeries were postponed. Approximately 400 RBC units containing WPM were inspected in the blood center and characterized into four types: I, II, III, and IV. A variety of preparations of aspirated WPM were made, including light and electron microscopic sections. RESULTS AND CONCLUSIONS: The rate of WPM-containing units was 1.67 percent (1 in 60 units), whereas the background incidence was less than 0.25 percent. Investigations revealed that WPM was composed of activated and nonactivated platelets (PLTs); no toxins, infectious agents, or agents of bioterrorism were identified. WPM correlated with certain variables studied, including PLT-rich components that had been centrifuged with a "hard spin" before leukoreduction and manufactured in one vendor's collection sets. Because the increased rate of appearance of WPM was a transient phenomenon, it is not clear whether this is a newly noticed or a new and different phenomenon from "aggregates" observed in the past.

Blood Specimen Collection↗
PubMed2004

Separating antiviral and GVHD activities of donor T cells prior to bone marrow transplantation.

Approaches to speed immune reconstitution following bone marrow transplantation (BMT) or peripheral-blood hematopoietic stem-cell transplantation (HSCT) could markedly reduce morbidity and mortality, particularly following partially major histocompatibility complex (MHC)-matched related donor (PMRD) transplants. However, it is critical to simultaneously eliminate the subpopulation of donor T cells that are alloreactive with the recipient and may produce graft-vs-host disease (GVHD). In this article, we discuss a number of promising cellular engineering approaches that could be applied to this problem, including the use of veto cells, regulatory T-cell subsets, and psoralen-treated donor lymphocytes. Emphasis is placed on whether these approaches can simultaneously transfer broad-spectrum immunity to the recipient without producing GVHD.

Adoptive Transfer↗
PubMed2003

Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation.

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 x 10(6) pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-gamma production by MCMV-primed CD8(+) T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log(10) and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44(high)) CD8(+) T cells persisted to day +100 following infusion, and expressed IFN-gamma when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.

Animals↗
PubMed2003

CMV DNA is rarely detected in healthy blood donors using validated PCR assays.

BACKGROUND: Although serologic screening or WBC reduction of blood components can reduce the incidence of transfusion-transmitted CMV (TT-CMV) infection, 'breakthrough' cases of TT-CMV still occur and may produce serious sequelae. NAT of blood components for CMV DNA has been proposed to further reduce the risks of TT-CMV. However, large-scale studies to determine the utility of validated CMV NAT assays for donor screening have not been reported. STUDY DESIGN AND METHODS: Coded whole-blood samples (n=1000) were tested for the presence of CMV DNA using two CMV PCR assays previously validated in a multicenter trial (a nested PCR assay directed at the CMV UL93 open-reading frame and the Roche Monitor assay). Corresponding plasma samples were tested in parallel for the presence of anti-CMV using other assays (Abbott CMV EIA and Fujirebio/Olympus CMV particle agglutination assays). RESULTS: In total 416 and 514 of the samples tested as CMV-seropositive and -seronegative, respectively, by both antibody assays. The remaining 70 samples had discrepant serology results. Only 2 of the 1000 samples (both seropositive) had reproducibly detectable CMV DNA (positive in at least three of four replicates). CMV DNA was not reproducibly detected in seronegative samples or in samples with discrepant serology results. CONCLUSIONS: Although previous investigations showed frequent detection of CMV DNA in healthy CMV-seropositive (and some seronegative) blood donors, these studies were relatively small and the performance characteristics of their assays were difficult to evaluate. In contrast, the present large cross-sectional study of US donors utilized two previously validated PCR assays and demonstrated that CMV DNA is only rarely detectable in seropositive donors. Thus, the use of CMV PCR assays with optimal performance characteristics did not increase the detection of potentially infectious blood components beyond that provided by current serologic screening assays.

Antibodies, Viral↗