Modeling the radiative effects of biomass burning aerosols on carbon fluxes in the Amazon region.

Every year, a dense smoke haze covers a large portion of South America originating from fires in the Amazon Basin and central parts of Brazil during the dry biomass burning season between August and October. Over a large portion of South America, the average aerosol optical depth at 550 nm exceeds 1.0 during the fire season, while the background value during the rainy season is below 0.2. Biomass burning aerosol particles increase scattering and absorption of the incident solar radiation. The regional-scale aerosol layer reduces the amount of solar energy reaching the surface, cools the near-surface air, and increases the diffuse radiation fraction over a large disturbed area of the Amazon rainforest. These factors affect the energy and CO2 fluxes at the surface. In this work, we applied a fully integrated atmospheric model to assess the impact of biomass burning aerosols in CO2 fluxes in the Amazon region during 2010. We address the effects of the attenuation of global solar radiation and the enhancement of the diffuse solar radiation flux inside the vegetation canopy. Our results indicate that biomass burning aerosols led to increases of about 27% in the gross primary productivity of Amazonia and 10% in plant respiration as well as a decline in soil respiration of 3%. Consequently, in our model Amazonia became a net carbon sink; net ecosystem exchange during September 2010 dropped from +101 to -104 TgC when the aerosol effects are considered, mainly due to the aerosol diffuse radiation effect. For the forest biome, our results point to a dominance of the diffuse radiation effect on CO2 fluxes, reaching a balance of 50-50% between the diffuse and direct aerosol effects for high aerosol loads. For C3 grasses and savanna (cerrado), as expected, the contribution of the diffuse radiation effect is much lower, tending to zero with the increase in aerosol load. Taking all biomes together, our model shows the Amazon during the dry season, in the presence of high biomass burning aerosol loads, changing from being a source to being a sink of CO2 to the atmosphere.

B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses.

A phase I-II trial to examine the toxicity of CMV- and EBV-specific cytotoxic T lymphocytes when used for prophylaxis against EBV and CMV disease in recipients of CD34-selected/T cell-depleted stem cell transplants.

Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV complications in recipients of CD34 selected/T-cell depleted stem cell transplants (SCT). EBV/CMV-specific CTL will be generated from peripheral blood mononuclear cells (PBMC) of EBV/CMV-seropositive donors in a course of from 21-28 days by weekly stimulation with autologous BLCLpp65. Qualified CTL will be administered to consenting patients at 40, 60, and 80 days post-transpOFF criteria of molecular virology and immunological reconstitution, which include blood levels of pp65 antigen and EBV viral DNA, and virus-specific CTL precursor frequency. Patients will also be tested for replication-competent retrovirus at 3, 6, and 12 month intervals post-transplant to ensure bio-safety.

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood.

Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.

Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation.

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell-depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/microg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had >/=40,000 copies of EBV DNA/microg DNA, all of whom were recipients of T-cell-depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with >/=40,000 copies EBV/microg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Outpatient total body irradiation for pediatric patients undergoing stem cell transplantation.

We have retrospectively reviewed the ability to safely deliver total body irradiation (TBI) in the outpatient setting in 10 pediatric patients undergoing stem cell transplantation. Patients had a median age of 14 years (range 9-17 years) with diagnoses that included ALL in second remission, AML in second remission, myelodysplastic syndrome, Ewing's sarcoma, and rhabdomyosarcoma. Patients received a total of 1375 cGy or 1440 cGy given in a hyperfractionated schedule (11 or 12 fractions) over a 4-day period. All children were seen in the outpatient clinic daily during TBI and all were housed within a 20 mile radius of our institution during this period. Eight patients achieved good control of nausea and emesis with ondansetron alone while two patients required ondansetron and diphenhydramine. Nine patients received some form of intravenous hydration during this period (hyperalimentation, fluid boluses in clinic, or night-time intravenous fluids). One patient maintained good hydration with oral intake alone. Only one child required admission during this period for persistent nausea and vomiting despite antiemetics and intravenous fluids. A cost approximation suggests that TBI delivered in the outpatient setting resulted in a saving of approximately $2400 per patient. We conclude that TBI administered to children and adolescents in the outpatient setting can be a safe and cost-effective practice.

Treatment of posttransplant lymphoproliferative disease in the central nervous system of a lung transplant recipient using allogeneic leukocytes.

Posttransplant Epstein-Barr virus-related lymphoproliferative disease (PT-LPD) is a common and often fatal complication following solid organ and hematopoietic stem cell transplantation. PT-LPD following solid organ transplantation generally occurs in B cells of recipient origin in contrast to PT-LPD in marrow transplant recipients, which is exclusively of donor origin. The efficacy of adoptive immunotherapy using donor leukocytes to treat PT-LPD in bone marrow transplant recipients has recently been reported. Because PT-LPD in solid organ transplant recipients is generally of recipient origin, the potential application of adoptive immunotherapy of PT-LPD in solid organ recipients obligates the use of either autologous or allogeneic HLA identical leukocytes, with the attendant risk of organ rejection if cells mismatched with the transplanted organ are used. Nonirradiated allogeneic mononuclear cells from an Epstein-Barr virus (EBV)-seropositive, HLA-identical normal sibling were used to treat a monoclonal EBV lymphoma of recipient origin in the central nervous system of a child who had undergone an HLA-mismatched cadaveric lung transplant. The patient received three separate mononuclear cell infusions over a 9-month period, each containing 1 x 10(6) CD3+ mononuclear cells per kilogram. Complete clinical, radiological, and pathological remission was achieved with this treatment regimen. The response correlated with in vivo reconstitution of normal EBV-specific cytotoxic activity and cytotoxic T lymphocyte precursor frequency. Use of allogeneic HLA-compatible mononuclear cells may thus offer an additional mode of therapy for EBV-related lymphoproliferative disease in selected solid organ transplant recipients refractory to conventional therapies.

Post-transplant EBV induced lymphoproliferative disorders.

Epstein Barr virus induced lymphoproliferative disease (EBV-LPD) is a heterogeneous disorder, ranging from polyclonal lymphoproliferations to malignant lymphoma, typically seen in individuals with inadequate cellular immunity to EBV. The diagnosis of EBV-LPD following transplant requires a high index of suspicion in those patients at risk. Unlike organ transplant patients, in whom lymphomas are generally of host origin and respond to decreases in immunosuppression, bone marrow transplant recipients have donor origin tumors that are not as responsive to conservative treatment modalities. For the latter group, adoptive immunotherapy with donor lymphocytes is the treatment of choice and generally results in complete eradication of these tumors. Whether adoptive immunotherapy can be used for EBV-LPD in patients following organ transplantation is currently being investigated.

Cord blood mononuclear cell transformation assay for screening for the presence of Epstein-Barr virus.

EBV-induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication following stem cell transplantation. Strategies have been developed for the cultivation of donor-derived, EBV-specific cytotoxic T lymphocytes (CTL) for stem cell transplant (SCT) patients affected with these disorders, using donor-derived, EBV-transformed B lymphoblastoid cell lines (BLCL) as stimulators. Although cultivation of EBV-transformed BLCL is possible without using an exogenous source of EBV, transformation of autologous B cells with endogenous virus may be slow and inconsistent. Therefore, if exogenous strains of EBV are used to generate BLCL, it may be beneficial to patients to ensure that these cell lines are not producing virus that potentially could be conveyed at the time of CTL infusion. A reliable method of screening for EBV using a cord blood transformation assay has been developed and is described.

The development of cellular immunity to Epstein-Barr virus after allogeneic bone marrow transplantation.

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a potentially lethal complication during the first 6 months after allogeneic bone marrow transplantation (BMT). To determine whether deficiencies of EBV-specific cellular immunity contribute to EBV-LPD susceptibility and distinguish patients at risk, we performed limiting dilution analysis to quantify anti-EBV cytotoxic T-lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or T-cell-depleted (TCD) grafts from EBV-seropositive donors. At 3 months post-BMT (n = 26), only five patients had EBV CTLp frequencies in the range of seropositive normal controls, irrespective of the type of transplant administered. By 6 months post-BMT, 9 of 13 patients tested had EBV CTLp frequencies within the normal range. The time period in which these patients had deficient cellular immunity to EBV corresponds to the period in which we have observed EBV-LPD in most prior patients. One patient with a low EBV CTLp frequency at 4 months post-BMT developed an EBV-LPD. Within 2 weeks of receiving an infusion of donor peripheral blood mononuclear cells (PBMC) providing less than 1,200 EBV-specific cytotoxic T-cell precursors, populations of EBV-specific CTL in the circulation were restored to levels detected in normal seropositive adults. Concurrently, the patient achieved a regression of the EBV-LPD, which has been sustained without further therapy. These studies indicate that recipients of both unmodified and TCD marrow grafts have profound deficiencies of EBV-specific T cell-mediated immunity early posttransplant, and that the period of risk for EBV-LPD closely corresponds to this interval of severe deficiency. Treatment of one patient with EBV-LPD with marrow donor-derived PBMC induced a rapid expansion of EBV-specific cytotoxic T-cell populations that occurred contemporaneously with the clinical regression of disease.

The identification of febrile, neutropenic children with neoplastic disease at low risk for bacteremia and complications of sepsis.

BACKGROUND: The management of pediatric oncology patients with fever and neutropenia assumes that all patients are at risk for bacteremia, and therefore generally involves hospitalization and broad-spectrum parenteral antibiotics for all patients. The determination of which patients are at low risk for having positive blood cultures and for developing complications related to bacteremia is of potential benefit. METHODS: The records of 161 pediatric patients with neoplastic disease hospitalized for 509 episodes of fever and neutropenia between January 1990 and June 1992 were retrospectively reviewed. Clinical features at initial presentation, clinical and microbiologic documentation of infection, and outcome were analyzed. RESULTS: The only presenting clinical features that correlated with an increased likelihood of having positive blood cultures were chills, hypotension, the requirement for fluid resuscitation (P < 0.001), or a diagnosis of leukemia or lymphoma (P < 0.041). Leukemia patients with relapse admitted for fever and neutropenia were no more likely to have positive blood cultures than those patients in remission. There were ten episodes of fever and neutropenia in which patients were transferred to the intensive care unit (ICU), and two sepsis related deaths. Nine episodes involving ICU management and both deaths were in patients who had persistent fever and an absolute neutrophil count (ANC) of less than 100 after 48 hours of hospitalization (n = 177). Patients with an ANC of less than 100 after 48 hours were twice as likely to have antibiotic changes, 15 times more likely to receive amphotericin B, and were hospitalized twice as long as patients with an ANC of 100 or higher after 48 hours. CONCLUSIONS: Children hospitalized for fever and neutropenia who have persistent fever and an ANC of less than 100 after 48 hours are at high risk for morbidity and are more likely to require antibiotic changes and antifungal therapy. Children who initially lack signs of sepsis, are afebrile, and have an ANC of 100 or higher after 48 hours are at low risk for complications and could be selectively discharged on antimicrobials after a 48-hour period of inpatient hospitalization.

Sepsis caused by Flavimonas oryzihabitans.

Previous reports of F. oryzihabitans sepsis involving central venous access devices reveal a relatively high rate of complications, including device removal, despite a course of broad-spectrum anti-microbials with compatible in vitro susceptibility results. In the present report of 22 cases of F. oryzihabitans sepsis treated at Memorial Sloan-Kettering Cancer Center from February 1986 through September 1993, the majority of CVAD-related infections with F. oryzihabitans were successfully treated with a 14-day course of antimicrobials with antipseudomonal activity, and removal of the device was usually not required. Factors that may complicate successful treatment of CVAD-related sepsis caused by F. oryzihabitans include polymicrobial infections and premature discontinuation of antibiotic therapy.

Cardiorespiratory decompensation following methylprednisolone administration.

A 2-year-old white female receiving multidrug chemotherapy for treatment of a primitive neuroectodermal tumor developed acute hypotension, bradycardia, and shock following administration of ondansetron and high-dose methylprednisolone. The subsequent clinical course is described, and cardiovascular reactions to ondansetron and methylprednisolone are reviewed. While the etiology of this severe reaction is uncertain, it is possible that it represents an idiosyncratic reaction to the rapid administration of high-dose adrenal corticosteroids. Patients receiving high-dose corticosteroid therapy should be closely monitored, and slow rates of infusion are recommended.