Adenovirus infection among pediatric patients with cancer and in pediatric recipients of hematopoietic stem cell: A multicenter nationwide study.

The aim was to evaluate the incidence, clinical course, and outcome of adenoviral infection (AdVI) in pediatric patients diagnosed and treated due to cancer and in pediatric recipients of hematopoietic stem cell. Over a 72-month period, all-in 5599 children with cancer: 2441 patients with hematological malignancy (HM) and 3158 with solid tumors (ST), and 971 patients after transplantation: 741 after allogeneic (allo-HSCT) and 230 after autologous (auto-HSCT) were enrolled into the study. Among cancer patients, 67 episodes of AdVI appeared in 63 (1.1%) children, including 45 (1.8%) with HM and 18 (0.6%; P < .001) with ST. Within transplanted patients, AdVIs were responsible for 88 episodes in 81 (8.3%) children (P < .001), including 78 (10.5%) patients after allo-HSCT and 3 (1.3%) after auto-HSCT. Time to develop AdVI was short, especially after allo-HSCT. The most common clinical manifestation in cancer patients was enteritis diagnosed in 63 (94.0%) cases, while among HSCT recipient asymptomatic adenoviremia was found in 36 (40.9%) cases and the most common clinical manifestation was urinary tract infection. Cancer patients with disseminated disease, as well as HSCT recipients with either asymptomatic viremia or disseminated disease, received antiviral treatment. The most commonly used first-line therapy was cidofovir. None of the cancer patients died due to AdVI, while within HSCT recipients three patients developed disseminated adenoviral disease and died despite antiviral treatment. In cancer patients, AdVIs are rare and associated with very good prognosis even without specific treatment. However, in allo-HSCT recipients, disseminated disease with fatal outcome is more likely to occur.

Hematopoietic stem cell transplantation does not increase the risk of infection-related complications for pediatric patients with Hodgkin and non-Hodgkin lymphomas: A multicenter nationwide study.

BACKGROUND: Hodgkin (HL) and non-Hodgkin lymphoma (NHL) represent a spectrum of lymphoid malignancies that are often curable with currently applied treatment regimens; however, 15%-30% of lymphoma patients still suffer from relapsed or refractory (rel/ref) disease. Although hematopoietic stem cell transplantation (HSCT) improves outcomes of second-line therapy for lymphoma in childhood, the complication rates in this group of patients, especially infectious complications (IC), remain unclear. OBJECTIVE: The aim of this population-based cohort study was a retrospective analysis of incidence, epidemiology and profile of bacterial infections (BI), invasive fungal disease (IFD), and viral infections (VI) in primary or rel/ref lymphoma patients, both HL and NHL. PATIENTS AND METHODS: We subdivided lymphoma patients into three groups: patients with primary conventional chemotherapy/radiotherapy regimens (group A), patients with rel/ref lymphoma treated with second-line chemotherapy (group B), and rel/ref lymphoma patients who underwent HSCT (group C). The medical records of the patients were biannually reported by each pediatric oncology center, and the data were analyzed centrally. RESULTS: Within 637 patients with primary lymphoma, at least one IC was diagnosed in 255 (40.0%), among 52 patients with rel/ref lymphoma 24 (46.2%) ICs were observed, and in transplanted group, 28 (57.1%) out of 49 children were diagnosed with IC (P = .151). The distribution of etiology of IC differed between the patient groups (A, B, C), with a predominance of BI in group A (85.6% vs 72.0% and 47.9%, respectively), VI in group C (9% and 16.0% vs 46.6%, respectively), and IFD in group B (5.4% vs 12.0% vs 5.5%, respectively). Overall, 500 (68.0%) episodes of bacterial IC were diagnosed in the entire group. Apart from HL patients treated with chemotherapy, in all the other subgroups of patients Gram-positives were predominant. The rate of multidrug-resistant bacteria was high, especially for Gram-negatives (41.1% in group A, 62.5% in group B, and 84.6% in group C). The infection-related mortality was comparable for each group. CONCLUSIONS: The incidence of IC was comparable during first- and second-line chemotherapy and after HSCT, but their profile was different for primary or re/ref lymphoma and depended on the type of therapy.

Multicenter Study of the Real-World Use of Ceftaroline versus Vancomycin for Acute Bacterial Skin and Skin Structure Infections.

The objective of this study was to determine if real-world ceftaroline treatment in adults hospitalized for acute bacterial skin and skin structure infections (ABSSSI) is associated with decreased infection-related length of stay (LOSinf) compared to that with vancomycin. This was a retrospective, multicenter, cohort study from 2012 to 2017. Cox proportional hazard regression, propensity score matching, and inverse probability of treatment weighting (IPTW) were used to determine the independent effect of treatment group on LOSinf The patients were adults hospitalized with ABSSSI and treated with ceftaroline or vancomycin for ≥72 h within 120 h of diagnosis at four academic medical centers and two community hospitals in Arizona, Florida, Michigan, and West Virginia. A total of 724 patients were included (325 ceftaroline treated and 399 vancomycin treated). In general, ceftaroline-treated patients had characteristics consistent with a higher risk of poor outcomes. The unadjusted median LOSinf values were 5 (interquartile range [IQR], 3 to 7) days and 6 (IQR, 4 to 8) days in the vancomycin and ceftaroline groups, respectively (hazard ratio [HR], 0.866; 95% confidence interval [CI], 0.747 to 1.002). The Cox proportional hazard model (adjusted HR [aHR], 0.891; 95% CI, 0.748 to 1.060), propensity score-matched (aHR, 0.955; 95% CI, 0.786 to 1.159), and IPTW (aHR, 0.918; 95% CI, 0.793 to 1.063) analyses demonstrated no significant difference in LOSinf between groups. Patients treated with ceftaroline were significantly more likely to meet criteria for discharge readiness at day 3 in unadjusted and adjusted analyses. Although discharge readiness at day 3 was higher in ceftaroline-treated patients, LOSinf values were similar between treatment groups. Clinical and nonclinical factors were associated with LOSinf.

Incidence, course, and outcome of Clostridium difficile infection in children with hematological malignancies or undergoing hematopoietic stem cell transplantation.

Clostridium difficile infection (CDI) is one of the most common causes of nosocomial infectious diarrhea in children during anticancer therapy or undergoing hematopoietic stem cell transplantation (HSCT) in Europe. Immunosuppression in these patients is a risk factor for CDI. Malignant diseases, age, acute graft-versus-host disease (aGVHD), HLA mismatch, or use of total body irradiation may play an important role in CDI course. The aim of this study was to evaluate the incidence, course, and outcome of CDI in children treated for malignancy or undergoing HSCT. Between 2012 and 2015, a total number of 1846 patients were treated for malignancy in Polish pediatric oncological centers (PHO group) and 342 underwent transplantation (HSCT group). In PHO group, episodes of CDI occurred in 210 patients (14%). The incidence of CDI was higher in patients with hematological malignancies in comparison to that with solid tumors. Patients with acute myeloblastic leukemia had shorter time to episode of CDI than those with acute lymphoblastic leukemia. Patients over 5 years and treated for acute leukemia had more severe clinical course of disease in PHO group. In HSCT group, CDI occurred in 29 (8%) patients. The incidence of CDI was higher in patients transplanted for acute leukemia. The recurrence rate was 14.7% in PHO and 20.7% in HSCT patients. CDI incidence was highest in patients with hematological malignancies. Most of patients experienced mild CDI. Age < 5 years and diagnosis other than acute leukemia were the positive prognostic factors influencing clinical CDI course.

Impact of High-Level Daptomycin Resistance in the Streptococcus mitis Group on Virulence and Survivability during Daptomycin Treatment in Experimental Infective Endocarditis.

Among the viridans group streptococci, the Streptococcus mitis group is the most common cause of infective endocarditis. These bacteria have a propensity to be ß-lactam resistant, as well as to rapidly develop high-level and durable resistance to daptomycin (DAP). We compared a parental, daptomycin-susceptible (DAPs) S. mitis/S. oralis strain and its daptomycin-resistant (DAPr) variant in a model of experimental endocarditis in terms of (i) their relative fitness in multiple target organs in this model (vegetations, kidneys, spleen) when animals were challenged individually and in a coinfection strategy and (ii) their survivability during therapy with daptomycin-gentamicin (an in vitro combination synergistic against the parental strain). The DAPr variant was initially isolated from the cardiac vegetations of animals with experimental endocarditis caused by the parental DAPs strain following treatment with daptomycin. The parental strain and the DAPr variant were comparably virulent when animals were individually challenged. In contrast, in the coinfection model without daptomycin therapy, at both the 106- and 107-CFU/ml challenge inocula, the parental strain outcompeted the DAPr variant in all target organs, especially the kidneys and spleen. When the animals in the coinfection model of endocarditis were treated with DAP-gentamicin, the DAPs strain was completely eliminated, while the DAPr variant persisted in all target tissues. These data underscore that the acquisition of DAPr in S. mitis/S. oralis does come at an intrinsic fitness cost, although this resistance phenotype is completely protective against therapy with a potentially synergistic DAP regimen.

First-line immunosuppressive treatment in children with aplastic anemia: rabbit antithymocyte globulin.

Immunosuppressive therapy is the treatment of choice in children with acquired severe aplastic anemia (AA) and no HLA-matched family donor. The paper presents results of a multicenter study of 63 children with AA treated with rabbit antithymocyte globulin (r-ATG) and cyclosporine A as the first line treatment in the years 1996-2012. Therapeutic effects were evaluated at Days 112, 180, and 360. At Day 112, remission was achieved in 28 out of the 63 patients (44.4 %), complete remission in 10 patients (15.9 %), and partial remission in 18 (28.5 %). At Day 180, 31 patients (49.2 %) were in remission including 15 cases in complete (23.8 %), and 16 cases in partial remission (25.4 %). One year after therapy onset, 34 patients (64.9 %) were in remission including 24 patients (38.0 %) in complete and 10 (15.9 %) in partial remission. Relapse occurred in 4 patients, from 8 months up to 2 years and 2 months after remission. One child, 5 years after remission, was diagnosed with paroxysmal nocturnal hemoglobinuria. The estimated 10-year overall survival rate and 10-year event-free survival rate were 67 % and 57 %, respectively.

Defining daptomycin resistance prevention exposures in vancomycin-resistant Enterococcus faecium and E. faecalis.

Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance (Dapr) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [Cmax] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [t1/2] of 8 h) were tested against one Enterococcus faecium strain (S447) and one Enterococcus faecalis strain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dapr. Mutations in genes encoding proteins associated with cell envelope homeostasis (yycFG and liaFSR) and phospholipid metabolism (cardiolipin synthase [cls] and cyclopropane fatty acid synthetase [cfa]) were investigated in Dapr derivatives. Dapr derivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Dapr after simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Dapr strains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations in liaF or cls. S447 derivatives lacked mutations in these genes. Dapr derivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT (P<0.01). Peak/MIC and AUC0-24/MIC ratios (AUC0-24 is the area under the concentration-time curve from 0 to 24 h) associated with Dapr prevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Dapr in serious enterococcal infections.

Novel combinations of vancomycin plus ceftaroline or oxacillin against methicillin-resistant vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA.

We demonstrated a significant inverse correlation between vancomycin and beta-lactam susceptibilities in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates. Using time-kill assays, vancomycin plus oxacillin or ceftaroline was synergistic against 3 of 5 VISA and 1 of 5 hVISA isolates or 5 of 5 VISA and 4 of 5 hVISA isolates, respectively. Beta-lactam exposure reduced overall vancomycin-Bodipy (dipyrromethene boron difluoride [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene] fluorescent dye) binding but may have improved vancomycin-cell wall interactions to improve vancomycin activity. Further research is warranted to elucidate the mechanism behind vancomycin and beta-lactam synergy.

The progressive reduction in the ovarian reserve in young women after anticancer treatment.

Anticancer treatment can disturb gonadal function and deplete the primordial follicle pool, leading to premature menopause. We made a prospective analysis of serum hormone levels in young female cancer survivors who had been treated during childhood and adolescence. Serum anti-Müllerian hormone (AMH) as a marker of ovarian reserve, FSH, LH, and estradiol were measured in 33 women treated previously (6-11 years earlier) for Hodgkin Lymphoma, solid tumours, and after bone marrow transplantation, and in 34 healthy controls. The group of survivors was divided according to the risk of gonadotoxicity into the low risk and median risk group (LR+MR), and into the high risk (HR) group. The measurements were repeated after 5 years. In the HR group, AMH levels were significantly lower than in controls (p=0.001) and in the LR+MR group (p=0.006) at the time of the first examination fell progressively after 5 years (p=0.03), whereas elevated FSH values (p=0.053) increased (p=0.001). Unchanged LH values in the first measurement rose in the second one (p=0.001). In the LR+MR group, the levels of AMH and FSH were normal (compared to the control) at baseline, but after 5 years serum AMH decreased (p=0.027) and FSH increased (p=0.008). Our findings indicate that anticancer treatment during childhood and adolescence is associated with a serious, progressive risk of ovarian failure. It is necessary to inform female cancer survivors, especially the high risk patients, about the risk of premature menopause.

Independent risk factors for the co-colonization of vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus in the region most endemic for vancomycin-resistant Staphylococcus aureus isolation.

In the majority of cases of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococcus faecalis (VR E. faecalis) served as the vanA donor to S. aureus. Previous studies that evaluated the risk factors for co-colonization with VRE and MRSA did not differentiate between VR E. faecalis and VR E. faecium. This study aimed to identify variables associated with VR E. faecalis and MRSA co-colonization. A retrospective case-control study from January 2008 to December 2009 was conducted at the Detroit Medical Center. Data were extracted from charts and pharmacy records. Unique patients co-colonized with VR E. faecalis and MRSA (defined as isolation of MRSA within 7 days of VR E. faecalis isolation) were compared with patients with VR E. faecalis who were not co-colonized with MRSA. A total of 546 patients with VR E. faecalis isolation were identified. 85 (15.6 %) VR E. faecalis patients were co-colonized with MRSA and 461 (84.4 %) VR E. faecalis patients were not co-colonized with MRSA. The mean age of the study cohort was 65.9 ± 16.4 years, 424 (77.7 %) were African-American, and 270 (49.5 %) were residing in long-term care institutions. Independent predictors of co-colonization of VR E. faecalis and MRSA were male gender, impaired consciousness, ICU stay prior to VR E. faecalis isolation, indwelling devices, and isolation of VR E. faecalis from wounds. MRSA was frequently isolated from the same culture specimen as VR E. faecalis (n = 39, 45.9 %), most commonly from wounds. This large study of patients with VR E. faecalis identified the severity of illness, indwelling devices, and chronic wounds as independent predictors of co-colonization with VR E. faecalis and MRSA.

Successful treatment of a left ventricular assist device infection with daptomycin non-susceptible methicillin-resistant Staphylococcus aureus: case report and review of the literature.

Recipients of left ventricular assist devices (LVADs) are highly susceptible to the development of infections with multidrug-resistant (MDR) organisms. We describe the case of a patient with an LVAD who developed a device-related, daptomycin non-susceptible, methicillin-resistant Staphylococcus aureus infection, highlighting this patient population as highly vulnerable to the development of such antimicrobial resistance. This report includes a thorough review of the literature on the mechanisms of development of daptomycin non-susceptibility and suggests ways to prevent its emergence. We also provide and underscore the appropriate guidelines to abide by when attempting to control infections with such resistant isolates. This case also demonstrates the importance of definitive treatment with LVAD removal and transplantation as a component of appropriate management of invasive LVAD infections. In addition, we suggest that even infections with MDR organisms may not adversely affect post-transplant outcomes.

Deshima 2.0: Rapid Redshift Surveys and Multi-line Spectroscopy of Dusty Galaxies.

We present a feasibility study for the high-redshift galaxy part of the Science Verification Campaign with the 220-440 GHz deshima 2.0 integrated superconducting spectrometer on the ASTE telescope. The first version of the deshima 2.0 chip has been recently manufactured and tested in the lab. Based on these realistic performance measurements, we evaluate potential target samples and prospects for detecting the [CII] and CO emission lines. The planned observations comprise two distinct, but complementary objectives: (1) acquiring spectroscopic redshifts for dusty galaxies selected in far-infrared/mm-wave surveys; (2) multi-line observations to infer physical conditions in dusty galaxies.

Cerebrospinal fluid IL-6, TNF-α and MCP-1 in children with acute lymphoblastic leukaemia during chemotherapy.

The aim of the study was to investigate the levels of cerebrospinal fluid (CSF) cytokines during chemotherapy of acute lymphoblastic leukaemia (ALL). Examination of 12 ALL child (6 boys and 6 girls) patients evidenced significant increases in interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) after induction treatment and significant increases in IL-6, tumour necrosis factor-α (TNF-α) and MCP-1 levels during the consolidation phase, as compared to their values at the time of diagnosis. There were no significant differences in CSF IL-6, TNF-α and MCP-1 concentrations after therapy. Our data suggest that standard ALL treatment may cause a subclinical inflammation and neurotoxicity.

The use of a sound-enabled device to measure pressure during insertion of an epidural catheter in women in labour.

The insertion of an epidural catheter for labour analgesia may be challenging. This observational study compared pressures during insertion of an epidural catheter in pregnant (n = 35) and non-pregnant (n = 10) women, using an acoustic device for locating the epidural space that also records and stores pressure data during the procedure. In both groups, we compared the maximum pressure just before loss of resistance, the pressure in the epidural space and the pressure in the inserted epidural catheter. Maximum pressure just before loss of resistance in the pregnant women was significantly lower compared with the non-pregnant women. Pressures in the epidural space and with the disposable tubing connected to the inserted epidural catheter were greater in pregnant women than in non-pregnant women. The results support the hypothesis that physiological changes in the third trimester of pregnancy are the reason why epidural catheters are more difficult to insert in women in labour.

Rediscovery of the enigmatic Andean frog Telmatobius halli Noble (Anura: Telmatobiidae), re-description of the tadpole and comments on new adult's characters, type locality and conservation status.

We report the rediscovery of Telmatobius halli (Hall's water frog), which had not been found since its description (over 80 years) since its type locality was not clearly established. "Aguas Calientes" near Ollagüe is hypothesized as the original type locality where Frank Gregory Hall collected the type material in 1935. The tadpole is re-described, and new data on the external and internal morphology of adults is provided. These new morphological data are compared with Telmatobius spp. inhabiting geographically close to T. halli in Chile and Bolivia. In addition, comments on its ecology, conservation, and taxonomic status in relation with other Telmatobius spp. inhabiting nearby areas in Ascotán and Carcote salt pans are provided. No evidence of Batrachochytrium dendrobatidis and Ranavirus infection was found in T. halli and a sympatric amphibian species. Our work supports the validity of T. halli and suggests this species should be considered as Data Deficient in the IUCN Red List assessment until taxonomic issues are resolved.

Cerebrospinal fluid changes in the excitatory amino acids concentration caused by the standard treatment of acute lymphoblastic leukaemia in children do not correlate with their later cognitive functioning.

The aim of this study was to ascertain whether changes in the concentrations of cerebrospinal fluid excitatory amino acids (EAAs) contribute to neurotoxicity of the standard acute lymphoblastic leukaemia (ALL) treatment protocols. We found a statistically significant increase in glutamate and aspartate in 12 ALL patients during their treatment. Cognitive functioning was examined in all patients at an average of 3.7 years after the disease diagnosis. Importantly, the levels of EAAs during the therapy were not correlated with the results of the cognitive test. This study suggests that standard ALL treatment-induced neurotoxicity may not lead to persistent neurocognitive deficits.

Acute lymphoblastic leukemia-derived dendritic cells express tumor associated antigens: PNPT1, PMPCB, RHAMM, BSG and ERCC1.

In all types of leukemia both in children and adults there is a need for novel therapies that could reduce the risk of relapse after standard treatment. Acute lymphoblastic leukemia (ALL) cells are ineffective antigen presenting cells, but as shown by many authors including results from our laboratory, stimulation with CD40L restores their antigen expressing capacity. The development of T-cell therapies for leukemic patients can be based on discovery of leukemia-associated antigens (LAA) which could be recognized by the host immune system. The aim of our present study was to test the hypothesis that leukemia-derived dendritic cells maintain the expression of tumor associated antigens. Twenty five children with B-cell precursor ALL were prospectively enrolled into the study. The mononuclear cells from peripheral blood or bone marrow were cultured and stimulated (or not) with CD40L and IL-4. The assessment of costimulatory/adhesion molecules with the use of flow cytometry and real-time RT PCR were used to confirm the possibility of turning ALL cells into dendritic-like cells. Additionally 22 tumor associated antigens mRNA levels were determined by real-time PCR technique with the TaqMan chemistry using ready-to-use Low Density Arrays for Gene Expression. The results of the study showed maintained expression and even up-regulation of some (PNPT1, PMPCB, HMMR/RHAMM, BSG and ERCC1) tumor associated antigens in CD40-activated leukemic cells. CD40L stimulation leading to the differentiation of leukemic cells into DCs which combine both antigen presenting function and expression of tumor associated antigens represents an interesting approach in cancer immunotherapy.

Genomic epidemiology of the emerging pathogen Batrachochytrium dendrobatidis from native and invasive amphibian species in Chile.

Emerging fungal diseases represent a threat to food security, animal and human health worldwide. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions. For the first time, Bd was cultured from native and non-native wild amphibians in Chile. Phylogenomic analyses revealed that Chilean isolates AVS2, AVS4 and AVS7 group within the global panzootic lineage of Bd (BdGPL) in a single highly supported clade that includes a genotype previously isolated from the United Kingdom. Our results extend the known distribution of BdGPL in South America and suggest a single and relatively recent introduction of BdGPL into the country, providing additional support to the role of anthropogenic activity in the global spread of this panzootic lineage.

Relationship between platelet secretion and prothrombin cleavage in native whole blood.

To determine the relationship between platelet secretion and prothrombin conversion in whole blood, the release of platelet factor 4 and the generation of a X(a)-specific cleavage product of prothrombin, fragment 1 + 2, were measured during the coagulation of whole blood. There was a parallel increase in the concentration of the two proteins. Over the first 5 min of incubation, platelet factor 4 concentration increased 6 ng/ml per min, and after 6-7 min, the rate of release increased to 750 ng/ml per min. Over the initial 5-7 min of incubation, fragment 1 + 2 concentration increased 1.5 pmol/ml per min with a subsequent increase of 45 pmol/ml per min. Incubation with 10 muM prostaglandin E(1) or 15 muM prostaglandin I(2) inhibited secretion of platelet factor 4 and delayed the onset of the rapid phase of fragment 1 + 2 generation by 8 min, while stimulation of platelet secretion with 1 mug/ml collagen suspension enhanced production of fragment 1 + 2. The addition of either 10 muM epinephrine or 100 ng/ml collagen suspension to whole blood did not affect either platelet factor 4 release or fragment 1 + 2 generation, although the combination of 3 muM epinephrine and 100 ng/ml collagen suspension enhanced platelet release and prothrombin cleavage. The relationship between platelet factor 4 release and prothrombin cleavage was also studied in Factor VIII-deficient blood. When 0.001 U/ml factor VIII activity was present, <80 ng/ml platelet factor 4 were released, and no fragment 1 + 2 was generated after 30 min of incubation. The addition of 0.008-0.08 U/ml Factor VIII activity progressively increased platelet factor 4 release and prothrombin cleavage. Platelet factor 4 release was normal at 0.08 U/ml Factor VIII activity, whereas prothrombin cleavage was still delayed. Very little thrombin, the amount generated by the cleavage of 3-5 nM fragment 1 + 2, was needed to induce release of platelet factor 4.