Baseline clinical characteristics and disease burden in patients with paroxysmal nocturnal hemoglobinuria (PNH): updated analysis from the International PNH Registry.

The International Paroxysmal Nocturnal Hemoglobinuria (PNH) Registry (NCT01374360) was initiated to optimize patient management by collecting data regarding disease burden, progression, and clinical outcomes. Herein, we report updated baseline demographics, clinical characteristics, disease burden data, and observed trends regarding clone size in the largest cohort of Registry patients. Patients with available data as of July 2017 were stratified by glycosylphosphatidylinositol (GPI)-deficient granulocyte clone size (< 10%, ≥ 10%-< 50%, and ≥ 50%). All patients were untreated with eculizumab at baseline, defined as date of eculizumab initiation or date of Registry enrollment (if never treated with eculizumab). Outcomes assessed in the current analysis included proportions of patients with high disease activity (HDA), history of major adverse vascular events (MAVEs; including thrombotic events [TEs]), bone marrow failure (BMF), red blood cell (RBC) transfusions, and PNH-related symptoms. A total of 4439 patients were included, of whom 2701 (60.8%) had available GPI-deficient granulocyte clone size data. Among these, median clone size was 31.8% (1002 had < 10%; 526 had ≥ 10%-< 50%; 1173 had ≥ 50%). There were high proportions of patients with HDA (51.6%), history of MAVEs (18.8%), BMF (62.6%), RBC transfusion (61.3%), and impaired renal function (42.8%). All measures except RBC transfusion history significantly correlated with GPI-deficient granulocyte clone size. A large proportion of patients with GPI-deficient granulocyte clone size < 10% had hemolysis (9.7%), MAVEs (10.2%), HDA (9.1%), and/or PNH-related symptoms. Although larger GPI-deficient granulocyte clone sizes were associated with higher disease burden, a substantial proportion of patients with smaller clone sizes had history of MAVEs/TEs.

Assessment of low-dose cisplatin as a model of nausea and emesis in beagle dogs, potential for repeated administration.

Cisplatin is a highly emetogenic cancer chemotherapy agent, which is often used to induce nausea and emesis in animal models. The cytotoxic properties of cisplatin also cause adverse events that negatively impact on animal welfare preventing repeated administration of cisplatin. In this study, we assessed whether a low (subclinical) dose of cisplatin could be utilized as a model of nausea and emesis in the dog while decreasing the severity of adverse events to allow repeated administration. The emetic, nausea-like behavior and potential biomarker response to both the clinical dose (70 mg/m2) and low dose (15 mg/m2) of cisplatin was assessed. Plasma creatinine concentrations and granulocyte counts were used to assess adverse effects on the kidneys and bone marrow, respectively. Nausea-like behavior and emesis was induced by both doses of cisplatin, but the latency to onset was greater in the low-dose group. No significant change in plasma creatinine was detected for either dose groups. Granulocytes were significantly reduced compared with baseline (P = 0.000) following the clinical, but not the low-dose cisplatin group. Tolerability of repeated administration was assessed with 4 administrations of an 18 mg/m2 dose cisplatin. Plasma creatinine did not change significantly. Cumulative effects on the granulocytes occurred, they were significantly decreased (P = 0.03) from baseline at 3 weeks following cisplatin for the 4th administration only. Our results suggest that subclinical doses (15 and 18 mg/m2) of cisplatin induce nausea-like behavior and emesis but have reduced adverse effects compared with the clinical dose allowing for repeated administration in crossover studies.

Efficacy, safety and cost analyses in ulcerative colitis patients undergoing granulocyte and monocyte adsorption or receiving prednisolone.

BACKGROUND: Patients with ulcerative colitis (UC) are treated with prednisolone (PSL), which causes adverse side effects. Extracorporeal granulocyte/monocyte adsorption (GMA) with an Adacolumn depletes elevated/activated myeloid lineage leucocytes as sources of inflammatory cytokines. We were interested to evaluate the efficacy, safety and the treatment cost for PSL and GMA. METHODS: Forty-one patients with active UC had achieved remission with GMA, at 1 or 2 sessions/week, up to 10 sessions (n=24) or with orally administered PSL (1mg/kg bodyweight, n=17). Clinical activity index (CAI) ≤4 was considered clinical remission. Following remission, patients received 5-aminosalicylic acid (2250-3000mg/day) or sulphasalazine (4000-6000mg/day) as maintenance therapy and were followed for 600 days. The total treatment cost was assessed based on 1€=150JPY. RESULTS: PSL was tapered after two weeks, and discontinued when a patient achieved remission. The average time to the disappearance of at least one major UC symptom (haematochezia, diarrhoea, or abdominal discomfort) was 15.3 days in the GMA group and 12.7 days in the PSL group, while time to remission was 27.9 days in the GMA group and 27.6 days in the PSL group, CAI 0.8 and 2.0, respectively. The Kaplan-Meier plots showed similar remission maintenance rates over the 600 days follow-up period. The average medical cost was 12739.4€/patient in the GMA group and 8751.3€ in the PSL group (P<0.05). In the GMA group, 5 transient adverse events were observed vs 10 steroid related adverse events in the PSL group (P<0.001). CONCLUSIONS: In appropriately selected patients, GMA has significant efficacy with no safety concern. The higher cost of GMA vs PSL should be compromised by good safety profile of this non-pharmacological treatment intervention.

Assessment of dysplastic hematopoiesis: lessons from healthy bone marrow donors.

BACKGROUND: According to WHO 2008 guidelines, the required percentage of cells manifesting dysplasia in the bone marrow to qualify as significant is 10% or over in one or more hematopoietic cell lineages, but this threshold is controversial. No 'normal' values have been established. Therefore, we investigated dyshematopoiesis in bone marrow aspirate squash preparations of 120 healthy bone marrow donors. DESIGN AND METHODS: Bone marrow squash slides of 120 healthy unrelated bone marrow donors were examined independently by 4 experienced morphologists. Samples were taken from the first aspiration during the harvest. Bone marrow preparation and assessment were performed according to WHO recommendations and ICSH guidelines. RESULTS: More than 10% dysmyelopoiesis could be detected in 46% of bone marrow aspirate squash preparations with 26% in 2 or more cell lineages and 7% in 3 cell lineages in healthy bone marrow donors. Donors under the age of 30 years exhibited more dysgranulopoietic changes and dysmegakaryopoietic changes (P<0.001) compared to the older donors. Female donors showed more dysgranulopoietic changes than male donors (P = 0.025). The concordance rate between the 4 investigators was modest in dysgranulopoiesis but poor in dyserythropoiesis and dysmegakaryopoiesis. CONCLUSIONS: The poor reliability of the 10% cut off was partly related to the proximity of the current criteria to the observed cut-off mean values of the normal population. These findings question the current WHO threshold of the 10% or over necessary for the percentage of cells manifesting dysplasia to be considered significant, and suggest that either a higher threshold would be more appropriate or different thresholds should be set for each lineage.

Intracellular detection of interleukin 17 and other cytokines in human bronchoalveolar lavage fluid: a first assessment.

Increasing evidence links pulmonary pathology to cytokines determining an inflammatory environment in the lung. Detection of cells secreting specific cytokines in BALF could be helpful as a diagnostic tool but which cytokines to choose among their great variety may be the first question to solve. The aim of this study was to investigate the Th1, Th2 and Th17 cytokine profile in whole cells within the human bronchoalveolar lavage fluid (BALF) by flow cytometry, with a focus on interleukin (IL)-17-producing cells, in order to assess which cytokines might lend themselves as markers of disease in future studies. BALF and paired peripheral blood samples were collected from 52 patients admitted to hospital for pulmonary pathologies. Cells obtained from BALF and peripheral blood were incubated in vitro in the absence or presence of appropriate stimuli and analyzed for intracellular content of IL-4, -10, -12, -17, interferon (IFN)γ and tumor necrosis factor (TNF)α in association to expression of either HLA-DR or CD4. IL-17-secreting cells were further characterized. Production of IL-17 by unstimulated BALF cells could be detected in 2 of the 32 patients that could be examined; upon PMA/IM stimulation in vitro, IL-17 was produced by varying percentages of lymphocytes, mostly memory CD4(+) cells, in all BALF samples. IL-4 could be detected in a relatively high proportion of unstimulated HLA-DR(+/-), SSC(hi) cells, most probably granulocytes; IL-10 could be found mostly in macrophages in a number of the BALF samples analyzed. Finally, IFNγ and TNFα were only produced by lymphocytes after in vitro stimulation. This study shows that T cells producing IL-17 can be found in the lung of respiratory patients in the absence of ex vivo stimulation, making IL-17 a good candidate marker of specific pathologies of the lung. Upon stimulation, IL-17 production was accounted for by CD4(+) CD45RO(+) cells. Other cytokines are also discussed. An interesting cytokine secretion profile found in BALF from a patient with rheumatoid lung disease is also reported.

The histological assessment of cutaneous vasculitis.

Vasculitis is defined as inflammation directed at vessels, which compromises or destroys the vessel wall leading to haemorrhagic and/or ischaemic events. Skin biopsy is the gold standard for the diagnosis of cutaneous vasculitis, whose manifestations include urticaria, infiltrative erythema, petechiae, purpura, purpuric papules, haemorrhagic vesicles and bullae, nodules, livedo racemosa, deep (punched out) ulcers and digital gangrene. These varied morphologies are a direct reflection of size of the vessels and extent of the vascular bed affected, ranging from a vasculitis affecting few superficial, small vessels in petechial eruptions to extensive pan-dermal small vessel vasculitis in haemorrhagic bullae to muscular vessel vasculitis in lower extremity nodules with livedo racemosa. Skin biopsy, extending to subcutis and taken from the earliest, most symptomatic, reddish or purpuric lesion is crucial for obtaining a high-yielding diagnostic sample. Based on histology, vasculitis can be classified on the size of vessels affected and the dominant immune cell mediating the inflammation (e.g. neutrophilic, granulomatous, lymphocytic, or eosinophilic). Disruption of small vessels by inflammatory cells, deposition of fibrin within the lumen and/or vessel wall coupled with nuclear debris allows for the confident recognition of small vessel, mostly neutrophilic vasculitis (also known as leukocytoclastic vasculitis). In contrast, muscular vessel vasculitis can be identified solely by infiltration of its wall by inflammatory cells. Extravasation of red blood cells (purpura) and necrosis are supportive, but not diagnostic of vasculitis as they are also seen in haemorrhagic and/or vaso-occlusive disorders (pseudovasculitis). Vasculitic foci associated with extravascular granulomas (palisaded neutrophilic and granulomatous dermatitis), tissue eosinophilia, or tissue neutrophilia signal the risk for, or co-existence of systemic disease. This essential histological information coupled with direct immunofluorescence and anti-neutrophil cytoplasmic data and clinical findings enables more precise and accurate diagnosis of localized and systemic vasculitis syndromes.

Quality assessment of heparin coatings by their binding capacities of coagulation and complement enzymes.

In vitro testing of blood contacting materials before clinical application is generally advisable. Four heparin coatings from different manufacturers were tested for adsorbed proteins and soluble activation markers. The surface with the highest antithrombin, thrombin, high-molecular-weight-kininogen (HMWK) and the lowest fibrinogen binding capacity (Carmeda, Medtronic) showed significantly lower levels of granulocytes and platelet activation (beta-TG, PMN-elastase release). No statistically significant differences in soluble markers of the coagulation system could be detected (F1 + 2, TAT). Interestingly, complement activation (TCC) was significantly reduced within the group of the lowest adsorption of the complement factor C3. Our data demonstrate that there is a relation between the binding affinity of proteins (C1-inhibitor, C3-complement) and the consecutive changes in complement activation (TCC). Therefore, measuring adsorbed proteins on artificial surfaces is a suitable, sensitive and very reproducible method for assessing the thrombogenicity of biomaterials.

Granulocyte accumulation in ischemic/reperfused myocardium: assessment with a technetium-99m-labeled antigranulocyte monoclonal antibody in the dog.

This study tested the usefulness of technetium-99m-labeled antigranulocyte monoclonal antibody BW250/183 (AGMAb) for identifying granulocyte accumulation in ischemic/reperfused canine myocardium. In dogs with 90 minutes coronary artery occlusion and 180 minutes reperfusion (n = 8), ischemic/reperfused myocardial samples demonstrated 8.5 +/- 2.4 times more Tc-99m-AGMAb accumulation than nonischemic samples. Dogs given Tc-99m-labeled nonspecific human immunoglobulin instead of Tc-99m-AGMAb (n = 3) had about half as much accumulation (4.5 +/- 1.6, P < .05). Ex vivo myocardial imaging of Tc-99m-AGMAb demonstrated marked uptake in infarcted regions identified by absent triphenyl tetrazolium chloride staining. The amount of uptake was inversely related to the severity of ischemia (determined by radioactive microspheres) and directly correlated with tissue myeloperoxidase activity, a specific marker of granulocyte accumulation. No increase in Tc-99m-AGMAb uptake occurred in dogs with 90 minutes ischemia and no reperfusion (n = 3) or 15 minutes ischemia and 180 minutes reperfusion (n = 2). In conclusion, Tc-99m-AGMAb is taken up in reperfused infarcted myocardium by both nonspecific and specific mechanisms. Because the amount of uptake reflects myocardial granulocyte accumulation, Tc-99m-AGMAb combined with nuclear imaging techniques may be useful for studying inflammatory processes in the heart in experimental animal models and human beings.

Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy.

Hematopoietic growth factors have been used to accelerate engraftment after bone marrow transplantation and to "mobilize" peripheral blood progenitor cells (PBPC). We report on the data in 85 consecutive patients with Hodgkin's disease who were treated in a single institution using different methods to obtain PB progenitor cells. Use of granulocyte colony-stimulating factor for mobilization resulted in a significantly accelerated time to recovery of granulocytes (10 days v 12 days, P < .01) when compared with "nonmobilized" PBPC recipients. Similarly, use of mobilized PBPC resulted in a significantly accelerated time to platelet engraftment (13 days v 30 days, P < .001) when compared with "nonmobilized" recipients. Moreover, there was a statistically significant difference in total costs in favor of the group receiving "mobilized" PBPC.

[Immunohistochemical assessment of survival from open skin wounds using paraffin sections]. / Immunhistochemischer Vitalitätsnachweis von offenen Hautwunden am Paraffinschnitt.

Investigation of vitality was done in paraffin-embedded tissue sections of open skin wounds by demonstration of a neutrophilic reaction marking the cells by naphthol AS-D chloroacetate esterase, by demonstration of enzyme activity by application of lysozyme antibodies and by demonstration of concentration of proteinase inhibitors by application of inhibitor antibodies using the indirect immunohistochemical peroxidase method. Survival time of the wounds could be determined in 10 cases (15-165 mins) and was unknown in 9 other cases of sudden death due to injury of the major vessels or heart. Postmortally inflicted injuries and cases of sudden death due to massive blunt trauma served as controls. In vital injuries, accumulation of proteinase inhibitors, particularly alpha-1-antichymotrypsin and alpha-2-macroglobulin, were demonstrable in the corium parallel to the wound surface. Protein accumulation was observed only sporadically in cases of sudden death and never in cases with postmortally inflicted wounds. Granulocytes and lysozyme were first observed in the corium after a survival time of more than 60 minutes.

Erythroid and myeloid maturation patterns related to progenitor assessment in the myelodysplastic syndromes.

Patterns of erythropoiesis and granulopoiesis were studied in 15 patients with myelodysplastic syndromes and in one with smouldering leukaemia by correlating CFU-C, BFU-E and CFU-E cloning efficiency with erythroid and myeloid maturation indices derived from quantitative 14C-autoradiography and with ferrokinetics. Maturation index of a cell lineage was defined as the ratio of cell production rate increase from the first to the last proliferative compartment over the corresponding normal value. The myeloid maturation index was reduced in all cases, but CFU-C progenitor frequency was increased by a factor of 3. Erythroid maturation index was also reduced in most cases, and BFU-E progenitor frequency was reduced by a factor of 2. Similarly, CFU-E cloning efficiency corrected for the erythroid maturation index was 2.5-10-fold lower than normal. Comparison of the erythroid maturation index with ferrokinetics revealed a constant ratio of ineffective erythropoiesis with a maturation disturbance in the proliferative pool twice that in the non-proliferative pool. These findings indicate basic qualitative and quantitative abnormalities and a difference in the patterns of production of erythroid and myeloid cells in the myelodysplastic syndromes: Myeloid progenitors are increased in number but their maturation is grossly abnormal. On the other hand, the apparent reduction in erythroid progenitors and the reduced erythroid maturation index are attributable to both premature cell death of the more mature erythroblasts and an increased proerythroblast proliferation, i.e. extra divisions in the proerythroblast compartment at the expense of maturation.

Detection and assessment of human tumours producing granulocyte-macrophage colony-stimulating factor (GM-CSF) by heterotransplantation into nude mice.

Production of granulocyte-macrophage colony-stimulating factor(s) (GM-CSF) by human tumours was investigated using heterotransplantation of a number of different tumours in nude mice. An increase in granulocyte numbers (> 20,000/mm3) in the peripheral blood of nude mice accompanied the growth of 9 of the 25 transplanted tumours. GM-CSF activity tested against normal human marrow cells was relatively high in 6 of these 9 tumours. Moreover there was either weak activity or none at all in 14 of the 16 tumours that failed to cause a definite granulocytosis. The correlation between granulocytosis and GM-CSF activity was 0.36, which was statistically significant (P < 0.01). These findings indicate that the transplantation of human tumours into nude mice can provide a useful tool for detection and characterization of granulopoietic factors derived from the tumours.

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.