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Objective: Polymorphonuclear cell functions frequently are impaired in critically ill patients, and restoration of normal functions could help to prevent nosocomial infections. The aim of this study was to evaluate the effects of pretreatment with granulocyte colony-stimulating factor (G-CSF) on bacterial pneumonia induced 48 hrs after peritonitis (cecal ligation and puncture [CLP]) in rats. Design: Controlled animal study. Setting: Research laboratory of an academic institution. Subjects: Male Sprague-Dawley rats. Interventions: First, the CLP model was characterized. Second, alveolar endotoxin instillation allowed us to evaluate the ability of neutrophils to migrate to airspaces after CLP was assessed. In the last set of experiments, CLP was followed by G-CSF treatment as a preventive therapy for subsequent bacterial superinfection induced by alveolar Pseudomonas aeruginosa instillation. Measurements and Main Results: CLP induced a brief increase in proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β) at the 6th hr followed by a longer-lived anti-inflammatory response (interleukin-10 increase from days 1 to 3) in plasma, compared with healthy rats. Impaired neutrophil migration to alveolar spaces denoting immunoparalysis was evidenced after endotracheal endotoxin instillation following CLP, compared with non-CLP rats challenged with endotoxin. No such impairment was found when G-CSF (100 μg/kg: glycosylated recombinant human G-CSF, Lenograstim) was given before endotoxin. G-CSF (100 μg/kg 24 and 48 hrs after CLP) given before endotracheal P. aeruginosa instillation increased bacterial clearance, as shown by P. aeruginosa counts in both bronchoalveolar lavage (8.9 x 10 3 ± 2.8 x 10 3 colony-forming units/mL vs. 3.3 x 10 4 ± 1.5 x 104 colony-forming units/mL with saline) and lung tissue (4.2 x 105 ± 1.0 x 105 colony-forming units/g vs. 1.5 x 10 6 ± 0.6 x 10 6 colony-forming units/g with saline). Furthermore, G-CSF pretreatment kept P. aeruginosa clearance in CLP rats similar to that in non-CLP rats challenged with P. aeruginosa. Conclusion: These results suggest that G-CSF (Lenograstim) may enhance host defenses in rats with peritonitis and immunoparalysis.

OBJECTIVE Granulocyte colony-stimulating factor is widely prescribed to hasten recovery from cancer chemotherapy-induced neutropenia and has been reported to induce pulmonary toxicity. However, circumstances and mechanisms of this toxicity remain poorly known. DESIGN To reproduce a routine situation in cancer patients receiving chemotherapy, we investigated the mechanisms underlying granulocyte colony-stimulating factor-induced exacerbation of alpha-naphthylthiourea-related pulmonary edema. SETTING Laboratory research unit. SUBJECTS Male specific-pathogen-free Sprague-Dawley rats. INTERVENTIONS The effects of granulocyte colony-stimulating factor given alone or after alpha-naphthylthiourea used to induce acute lung injury were investigated. MEASUREMENTS AND MAIN RESULTS Lung injury was assessed based on neutrophil sequestration (myeloperoxidase activity in lung tissue) and influx into alveolar spaces (bronchoalveolar lavage fluid cell quantification) and on edema formation (wet/dry lung weight ratio) and alveolar protein concentration into bronchoalveolar lavage fluid. Tumor necrosis factor-alpha and interleukin-1beta were measured in serum, lung homogenates, and isolated alveolar macrophage supernatants. In control rats, granulocyte colony-stimulating factor (25 microg/kg) significantly elevated circulating neutrophil counts without producing alveolar recruitment or pulmonary edema. alpha-Naphthylthiourea significantly increased the wet/dry lung weight ratio (4.68 +/- 0.04 vs. 4.38 +/- 0.07 in controls, p=.04) and induced alveolar protein leakage. Adding granulocyte colony-stimulating factor to alpha-naphthylthiourea exacerbated pulmonary edema, causing neutrophil sequestration in pulmonary vessels, significantly increasing lung myeloperoxidase activity (12.7 +/- 2.0 mOD/min/g vs. 1.1 +/- 0.4 mOD/min/g with alpha-naphthylthiourea alone; p

Background. Chimerism analysis is essential in understanding the etiology of graft failure occurring after allogeneic stem cell transplantation. The detection of marrow and/or blood host cells suggests graft rejection, relapse of the underlying disease, or a state of stable mixed chimerism. However, complete donor chimerism may be observed in some cases. Our objective was to characterize, by a sensitive process of chimerism analysis, six cases of graft failure occurring after transplant. Methods. Six cases of secondary graft failure, in which previous analysis had shown complete donor chimerism by standard polymerase chain reaction amplification of variable number of tandem repeats, were studied. In order to detect a minority population of recipient cells, we increased the sensitivity of the process by using fluorescent polymerase chain reaction and analyzing the origin of T, B, and natural killer lymphocytes at the time of graft failure. Results. The complete donor origin of mononuclear cells and lymphocytic populations was confirmed with this method in five of six patients. In the remaining patient, diagnosis of graft failure was clarified by the detection of a previously undetected mixed chimerism, compatible with graft rejection. In the other five patients, graft rejection was thereby excluded and graft failure could be related to viral infection or to graft-versus-host disease. Conclusion. Our sensitive process of fluorescent lineage-specific chimerism analysis may help in distinguishing between graft rejection and other mechanisms of graft failure, which is essential for deciding appropriate therapy. Secondary graft failure, the occurrence of pancytopenia after initial engraftment of an allogeneic transplant, is often a diagnostic problem. Recipients of T cell-depleted marrow may be particularly at risk for graft failure (1, 2), but it may be seen in recipients of unmodified, HLA-identical or nonidentical, marrow (3, 4). Immunologically mediated graft rejection, secondary lack of sustained marrow engraftment, and relapse of the underlying disease are the main diagnoses to be considered in this situation. Chimerism analysis is necessary to distinguish among these possibilities, if relapse is not apparent in marrow morphology. In some cases, the detection of residual host cells by chimerism analysis may represent a small number of malignant host cells not evident on marrow slides (5, 6). In other cases, the detection of residual host cells suggests either graft rejection (7) or a state of long-term stable mixed chimerism (8). To distinguish between these possibilities, the degree and evolution of mixed chimerism, determined by serial analysis over time, may be helpful. In contrast, the persistence of complete donor chimerism favors secondary lack of engraftment, as a result of infection (especially cytomegalovirus), the toxic effect of drugs, or graft-versus-host disease (GVHD) (9, 10). The sensitivity of detection of recipient cells is the essential parameter of chimerism analysis in this setting (11). A minority population of recipient cells could be undetected against a background of donor cells if an insensitive process is used. The suitable process of chimerism analysis must be applicable to the small number of cells that can be collected from hypocellular blood or marrow at the time of graft failure. Our objective was to study cases of secondary graft failure with a sensitive method of chimerism analysis. We retrospectively studied six patients, in whom previous analysis had shown complete donor chimerism using standard polymerase chain reaction (PCR) amplification of variable number of tandem repeat (VNTR) sequences. In order to detect a minority population of recipient cells, we sought to increase the sensitivity of the process by using fluorescent primers and automated GeneScan analysis of PCR products. In five patients, we applied this method separately to lymphocytic T-, B-, and natural killer (NK) cell subpopulations, sorted at the time of graft failure. We also examined the potential etiologies of graft failure and the impact of therapy on achieving subsequent engraftment.

Twenty patients (17 women, three men) with mediastinal diffuse large-cell lymphoma with sclerosis are reported. At the time of diagnosis, the disease was confined to supradiaphragmatic areas in all patients but two, who had kidney involvement (seven were stage I, 11 were stage II, and two were stage IV). A B-cell phenotype was demonstrated in nine of the 11 cases that were analyzed for cell lineage. All patients received an anthracyclin-containing regimen followed by mantle radiotherapy. Analysis of pretreatment characteristics showed that only the extent of disease influences outcome. Moreover, achievement of complete remission (CR) after chemotherapy appears to be a major prognostic factor. Two-year survival was better in patients who reached CR after chemotherapy than in those who did not (100% versus 9%; p less than 0.0001). Overall 2-year and 7-year survival rates were 50% and 33%, respectively. Therefore, localized mediastinal large cell lymphoma with sclerosis should be considered a high-risk subtype of non-Hodgkin's lymphoma in which standard treatment approaches are unsuccessful.

Twenty-two patients with Stage III and IV follicular non-Hodgkin's lymphoma were treated for 8 months by a chemotherapy regimen alternating between two different four-drug combinations (doxorubicin, teniposide, cyclophosphamide, prednisone; vincristine, cyclophosphamide, CCNU, prednisone). The overall response rate (complete and partial remission) was 68%. The complete remission rate was 23%. Twenty patients are alive, 50% of them are free of disease progression (median follow-up 76 months). It is concluded that our chemotherapy regimen is not satisfactory and that new therapeutic approaches are needed.