A prospective observational study of the rapid detection of clinically-relevant plasma direct oral anticoagulant levels following acute traumatic injury.

In urgent clinical situations, such as trauma, urgent surgery or before thrombolysis, rapid quantification of direct oral anticoagulant plasma drug levels is warranted. Using the ClotPro® analyser, we assessed two novel viscoelastic tests for detection of clinically-relevant plasma drug levels in trauma patients. The ecarin clotting time was used to assess the plasma concentration of dabigatran and Russell´s viper venom clotting time to determine the plasma concentration of direct factor Xa inhibitors. In parallel, plasma concentrations were analysed using plasma-based chromogenic assays. A total of 203 simultaneous measurements were performed. Strong to very strong linear correlations were detected between ecarin clotting time and plasma concentration of dabigatran (r = 0.9693), and between Russell´s viper venom clotting time and plasma concentrations of apixaban (r = 0.7391), edoxaban (r = 0.9251) and rivaroxaban (r = 0.8792), all p < 0.001. An ecarin clotting time ≥ 189 seconds provided 100% sensitivity and 90% specificity for detecting plasma dabigatran concentrations ≥ 50 ng.ml-1 . Corresponding Russell´s viper venom clotting time cut-off values were ≥ 136 seconds for apixaban (80% sensitivity, 88% specificity), ≥ 168 seconds for edoxaban (100% sensitivity, 100% specificity) and ≥ 177 seconds for rivaroxaban (90% sensitivity, 100% specificity). Detection of drug levels ≥ 100 ng.ml-1 was also investigated: for dabigatran, an ecarin clotting time ≥ 315 seconds yielded 92% sensitivity and 100% specificity; while Russell´s viper venom clotting time cut-offs of 191, 188 and 196 seconds were calculated for apixaban (67% sensitivity, 88% specificity), edoxaban (100% sensitivity, 75% specificity) and rivaroxaban (100% sensitivity, 91% specificity), respectively. We have demonstrated strong positive correlations between plasma drug levels and clotting time values in the specific ClotPro assays. Cut-off values for detecting clinically-relevant drug levels showed high levels of sensitivity and specificity.

Relationships between early-life growth, intake, and birth season with first-lactation performance of Holstein dairy cows.

The objective was to determine the relationships between early-life parameters [including average daily gain (ADG), body weight (BW), milk replacer intake, starter intake, and birth season] and the first-lactation performance of Holstein cows. We collected data from birth years 2004 to 2012 for 2,880 Holstein animals. Calves were received from 3 commercial dairy farms and enrolled in 37 different calf research trials at the University of Minnesota Southern Research and Outreach Center from 3 to 195 d. Upon trial completion, calves were returned to their respective farms. Milk replacer options included varying protein levels and amounts fed, but in the majority of studies, calves were fed a milk replacer containing 20% crude protein and 20% fat at 0.57 kg/calf daily. Most calves (93%) were weaned at 6 wk. Milk replacer dry matter intake, starter intake, ADG, and BW at 6 wk were 21.5 ± 2.2 kg, 17.3 ± 7.3 kg, 0.53 ± 0.13 kg/d, and 62.4 ± 6.8 kg, respectively. Average age at first calving and first-lactation 305-d milk yield were 715 ± 46.5 d and 10,959 ± 1,527 kg, respectively. We conducted separate mixed-model analyses using the REML model-fitting protocol of JMP (SAS Institute Inc., Cary, NC) to determine the effect of early-life BW or ADG, milk replacer and starter intake, and birth season on first-lactation 305-d milk, fat, and true protein yield. Greater BW and ADG at 6 wk resulted in increased first-lactation milk and milk component yields. Intake of calf starter at 8 wk had a significant positive relationship with first-lactation 305-d yield of milk and milk components. Milk replacer intake, which varied very little in this data set, had no effect on first-lactation 305-d yield of milk and milk components. Calves born in the fall and winter had greater starter intake, BW, and ADG at 8 wk. However, calves born in the summer had a higher 305-d milk yield during their first lactation than those born in the fall and winter. Improvements were modest, and variation was high, suggesting that additional factors not accounted for in these analyses affected first-lactation performance.

Randomized crossover study assessing oropharyngeal leak pressure and fiber optic positioning : Laryngeal Mask Airway Supreme™ versus Laryngeal Tube LTS II™ size 2 in non-paralyzed anesthetized children.

BACKGROUND: As there are currently no data available comparing the practicability of the laryngeal mask airway (LMA) Supreme™ size 2 versus the laryngeal tube LTS II™ size 2 in children, this trial was conducted to quantify the differences between these two airway devices concerning leak pressure and fiber optic-controlled positioning in non-paralyzed, anesthetized pediatric patients. METHODS: A total of 56 children aged 1-6 years and weighing between 11 and 23 kg were enrolled in the study. Anesthesia was intravenously induced according to local standards using fentanyl and propofol. After induction of anesthesia both airway devices were inserted consecutively in accordance with the randomization protocol. RESULTS: The mean oropharyngeal leak pressure was significantly higher for the LTS II™ (33±8 cmH2O) than for the LMA Supreme™ (21±7 cmH2O, p < 0.0001). Fiber optic position monitoring was better when the LMA Supreme™ was used (p  < 0.001). The first attempt success rates for insertion (55Supreme LMA vs. 43LTSII, p < 0.001), the insertion time (25 s Supreme LMA vs. 34 s LTSII, p < 0.04) and the frequency of bloodstaining (0Supreme LMA vs 4LTSII, p < 0.04) for the initially used device were better for the LMA Supreme™ than the laryngeal tube LTS II™. CONCLUSION: We conclude that oropharyngeal leak pressure, fiber optic position, first attempt insertion success rate and bloodstaining differed between the LMA Supreme™ and the LTS II™ in children.

Domain wall roughness in stripe phase BiFeO3 thin films.

Using the model system of ferroelectric domain walls, we explore the effects of long-range dipolar interactions and periodic ordering on the behavior of pinned elastic interfaces. In piezoresponse force microscopy studies of the characteristic roughening of intrinsic 71° stripe domains in BiFeO3 thin films, we find unexpectedly high values of the roughness exponent ζ=0.74±0.10, significantly different from those obtained for artificially written domain walls in this and other ferroelectric materials. The large value of the exponent suggests that a random field-dominated pinning, combined with stronger disorder and strain effects due to the step-bunching morphology of the samples, could be the dominant source of pinning in the system.

Effects of an early rehabilitation program for adult cystic fibrosis patients during hospitalization: a randomized clinical trial.

There is little information on pulmonary rehabilitation in patients with cystic fibrosis (CF) with pulmonary exacerbation. This study aimed to evaluate the effects of an early rehabilitation program on lung function, muscle strength, inflammatory markers, and quality of life in adults with CF hospitalized for pulmonary exacerbation. In this randomized controlled trial, 19 patients were included in the intervention group and 15 in the control group. The intervention group underwent an early rehabilitation program for 14 days after admission. All patients underwent spirometry, one-repetition maximum tests (1RM), and the 6-min walk test, and answered the Revised Cystic Fibrosis Questionnaire (CFQ-R) for quality of life and the International Physical Activity Questionnaire. Serum levels of interleukin and tumor necrosis factor alpha (TNF-α) were measured. In the intervention group, there were increases in 1RM biceps (P=0.009), triceps (P=0.005), shoulder abductors (P=0.002), shoulder flexors (P=0.004), hamstrings (P<0.001), and quadriceps values (P<0.001). In addition, there were improvements in CFQ-R-emotion (P=0.002), treatment burden (P=0.002), vitality (P=0.011), and physical scores (P=0.026), and a reduction in the Borg resting fatigue score (P=0.037). The interleukins levels did not change after the intervention. In adult CF patients with pulmonary exacerbation, early hospital rehabilitation had a significant impact on improving resting fatigue, muscle strength, and quality of life.

[Anesthesiological aspects in patients with epidermolysis bullosa]. / Anästhesiologische Aspekte bei Patienten mit Epidermolysis bullosa.

Epidermolysis bullosa causes blistering due to altered structural proteins of the dermoepidermal junction, resulting in scarring and strictures of the skin and mucous membranes. Affected individuals typically require frequent surgical interventions due to burdensome symptoms and complications of the disease. The anesthesiological management of these patients is inherently challenging. This review article summarizes the relevant features of this patient cohort and provides practical recommendations for care.

Functional capacity, lung function, and muscle strength in patients undergoing hematopoietic stem cell transplantation: A prospective cohort study.

OBJECTIVE/BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is a treatment for benign and malignant hematological diseases. These aggressive treatments cause reduced levels of physical activity, decreased lung function, and worse quality of life. Alterations in pulmonary function tests before HSCT are associated with the risk of respiratory failure and early mortality. The objective of this study was to evaluate functional capacity and lung function before and after HSCT and identify the predictors of mortality after 2 years. METHODS: A prospective cohort study was carried out with individuals with oncohematological diseases. The evaluations were carried out in two moments during hospitalization and at hospital discharge. Follow-up was carried out after 48 months. Assessments were carried out on 34 adults, using spirometry, manovacuometry, 6-Minute Walk Test (6MWT), Handgrip Strength Test, and 30-Second Chair Stand Test (30-s CST). RESULTS: There was a statistically significant reduction for the variables in forced vital capacity, forced expiratory volume predicted in the 1st second, Tiffeneau index, handgrip strength, and distance covered (% predicted) on the 6MWT (p < .05). There was a significant difference in the 30-s CST when individuals were compared according to the type of transplant. We found that a 10% reduction in the values of maximum inspiratory pressure (MIP) can predict an increased risk for mortality. CONCLUSIONS: Individuals undergoing HSCT have reduced functional capacity, lung function, and muscle strength during the hospitalization phase. Reduction in the values of MIP increases the risk of nonrelapse mortality.

Random-pattern versus perforator-based adipocutaneous skin paddles for postoperative monitoring of free muscle flaps-a comparative retrospective cohort study.

INTRODUCTION: Free microvascular muscle flaps represent well-established reconstructive options for complex soft tissue defects. However, due to their lack of cutaneous capillary beds, they are difficult to monitor postoperatively. To this end, random and axial-pattern adipocutaneous skin paddles are often included. The objective of the study was to compare the impact of random-pattern versus perforator-based adipocutaneous skin paddles on operative efficacy and muscle flap safety. METHODS: Between August 2014 and July 2016, a total of 120 free muscle flaps were included in this retrospective monocentric cohort study. Based on their skin-paddle type, they were either grouped into a 'perforator-based' (group Pb) or 'random-pattern' (group Rp) cohort. The electronic medical records and operative reports of all patients were subsequently reviewed and patient, defect, and flap characteristics of both groups were compared. The effect of the competing skin paddle types on the overall operative time, incidences of flap loss or microvascular complications, and total length of hospital stay were then assessed. RESULTS: Group Pb comprised 72 flaps, whereas 48 flaps constituted group Rp. Patient, defect, and flap characteristics were similar between both groups. Groups Pb and Rp were comparable regarding patient age (group Pb: 61 (10-90) vs. Rp: 59 (13-81), p = 0.556), ASA (American Society of Anesthesiologists) class (group Pb: 3 (1-4) vs. Rp 3 (1-3), p = 0.977), and comorbidities, summarized by the Charlson comorbidity index (CCI; group Pb: 1 (0-4 vs. Rp: 1 (0-5), p = 0.295). Both types of monitoring skin paddles were equally reliable. There was no significant difference in the mean operation time between both groups (group Pb: 373 ±â€¯122 min vs. Rp: 342 ±â€¯84 min, p = 0.124). In-patient treatment after flap surgery and total length of hospital stay were significantly shorter in group Pb (group Pb: 24 ±â€¯10 days vs. Rp: 32 ±â€¯17 days, p = 0.002 and group Pb: 39 ±â€¯15 vs. Rp: 48 ±â€¯24, p = 0.022). CONCLUSION: Perforator-based skin paddles are a reliable tool for postoperative perfusion monitoring of free muscle flaps and help avoid additional surgical interventions as opposed to their random-pattern counterparts. Thus, the overall and postoperative length of hospital stay is significantly reduced.

[Vaccination status in very and extremely preterm infants at the age of 2 years--a nationwide pilot analysis]. / Impfstatus bei Frühgeborenen mit einem Geburtsgewicht unter 1500 g im Alter von 2 Jahren--eine deutschlandweite Piloterhebung.

Preterm infants are at increased risk of suffering from vaccine preventable diseases. Therefore it is recommended to vaccinate them in time at the same chronological age as full term infants. Several reports have shown that vaccinations of very and extreme low birth weight infants are often delayed but till now there are no data for Germany. Our study analysed the vaccination rates and suffered complications of 318 preterm infants with birth weights under 1 500 g at the age of 2 years from all over the country copying their vaccination-record and asking the parents to answer a questionnaire. The preterm infants received the first 3 doses of the pentavalent or hexavalent vaccines consequently (1st dose: 100%, 2nd dose: 100%, 3rd dose: 97.5%), whereas the booster dose was accomplished to a lesser extent (89.0%) during their second year of life. The majority of the vaccinations were administered delayed. Moreover, the preterm infants received the first doses of the pneumococcal conjugate vaccine and the measles-mumps-rubella vaccine quite consequently, but the booster dose was again injected less frequent. According to their parents 17.8% of these preterm infants suffered from non-severe adverse reactions and none of them had an episode of apnoea following the immunisation. In conclusion preterm infants in Germany receive their basic immunisations consequently, but often too late. There is still the need to improve the frequency of the booster dose application especially in the second year of life and to prevent delays of vaccinations.

Evidence of stage progression in a novel, validated fluorescence-navigated and microsurgical-assisted secondary lymphedema rodent model.

Secondary lymphedema (SL)is a frequent and devastating complication of modern oncological therapy and filarial infections. A lack of a reliable preclinical model to investigate the underlying mechanism of clinical stage progression has limited the development of new therapeutic strategies. Current first line treatment has shown to be merely symptomatic and relies on lifetime use of compression garments and decongestive physiotherapy. In this study, we present the development of a secondary lymphedema model in 35 rats using pre- and intraoperative fluorescence-guided mapping of the lymphatics and microsurgical induction. In contrast to the few models reported so far, we decided to avoid the use of radiation for lymphedema induction. It turned out, that the model is nearly free of complications and capable of generating a statistically significant limb volume increase by water displacement measurements, sustained for at least 48 days. A translational, accurate lymphatic dysfunction was visualized by a novel VIS-NIR X-ray ICG-Clearance-Capacity imaging technology. For the first-time SL stage progression was validated by characteristic histological alterations, such as subdermal mast cell infiltration, adipose tissue deposition, and fibrosis by increased skin collagen content. Immunofluorescence confocal microscopy analysis suggested that stage progression is related to the presence of a characteristic α SMA+/HSP-47+/vimentin+ fibroblast subpopulation phenotype. These findings demonstrate that the in-vivo model is a reliable and clinically relevant SL model for the development of further secondary lymphedema therapeutic strategies and the analysis of the veiled molecular mechanisms of lymphatic dysfunction.

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

Impact of conventional or intensive milk replacer programs on Holstein heifer performance through six months of age and during first lactation.

The objectives were to evaluate the impact of conventional or intensive milk replacer (MR) feeding programs on heifer calf performance through 6 mo of age, age at first calving, and first lactation performance. At 3 (+/-1 d) d of age, 133 Holstein heifer calves from 3 commercial dairy farms were randomly assigned, within calf source, to a conventional [20% crude protein (CP), 20% fat] or intensive MR (28% CP, 18% fat). Milk replacer treatments and percent solids were 1) conventional nonacidified (CNA), 13.9%; 2) conventional acidified (CA), 13.9%; 3) modified intensive high solids (IHS), 16.7%; 4) modified intensive low solids (ILS), 12.5%; and 5) intensive high solids, high feeding (IHSHF), 16.7%. Calves were individually housed and remained on trial for 56 d. At 2 mo of age, heifers were grouped in pens by treatment with 6 heifers per pen (4 pens per treatment). An 18.1% CP grower concentrate mix (dry matter basis) was fed to heifers that received a conventional MR and a 21.2% CP grower concentrate mix was fed to heifers that received the intensive MR preweaning. Heifers were offered 2.45 kg/d (dry matter basis) of their respective grower concentrate mix for 112 d plus free access to hay and water. At approximately 24 wk of age, heifers were transported to a second-stage grower before returning to their respective farms approximately 1 mo before calving. First-lactation performance was determined using Dairy Herd Improvement Association records. The IHSHF treatment resulted in increased calf body weight and hip height during the preweaning and early postweaning (PEP) period and the postweaning heifer grower (PHG) period as compared with the conventional (CNA and CA) or modified intensive MR treatments (IHS and ILS). Calves receiving the IHS treatment were heavier at d 56 of the PEP period compared with the conventional or ILS treatments; however, this growth advantage was not maintained in the PHG period. Feed cost per kilogram of gain during the PEP period was lowest for CNA and CA, intermediate for IHS and ILS, and highest for the IHSHF treatment. There was no effect of MR feeding program on first-lactation performance; however, heifers that received the IHSHF MR preweaning calved 27.5 d earlier than those fed a conventional MR.

Detection of osteoarthritis using acoustic emission analysis.

Osteoarthritis (OA) of the knee is a widespread disease, often resulting in pain, restricted mobility and a reduction of activities and participation. Initial studies gave hints that Acoustic Emission Analysis (AEA) is capable of detecting early changes in cartilage structure. However, up to date no in vivo validation studies have been conducted. A prospective pilot study was conducted to investigate this diagnostic capability and the accuracy of the AEA, using magnetic resonance imaging (MRI) as a reference standard. Additionally, potential factors influencing false positive or negative results were studied. Twenty-eight patients, receiving MRI due to discomfort of the knee, were examined with AEA. Sensitivity was 0.92 for the whole knee and 0.86 to 1 for different parts of the knee. The specificity was 0.7 and 0.59 to 0.78, respectively. Confidence intervals varied between 0 and 0.33 for sensitivity and 0.1 and 0.24 for specificity. The diagnostic accuracy of the AEA was shown to be good to very good. However, because of the relatively small number of patients involved, interpretation of the data should be handled with care. Future studies with greater sample sizes have to be conducted to confirm the results of this investigation.

Effects of an early rehabilitation program for adult cystic fibrosis patients during hospitalization: a randomized clinical trial

There is little information on pulmonary rehabilitation in patients with cystic fibrosis (CF) with pulmonary exacerbation. This study aimed to evaluate the effects of an early rehabilitation program on lung function, muscle strength, inflammatory markers, and quality of life in adults with CF hospitalized for pulmonary exacerbation. In this randomized controlled trial, 19 patients were included in the intervention group and 15 in the control group. The intervention group underwent an early rehabilitation program for 14 days after admission. All patients underwent spirometry, one-repetition maximum tests (1RM), and the 6-min walk test, and answered the Revised Cystic Fibrosis Questionnaire (CFQ-R) for quality of life and the International Physical Activity Questionnaire. Serum levels of interleukin and tumor necrosis factor alpha (TNF-α) were measured. In the intervention group, there were increases in 1RM biceps (P=0.009), triceps (P=0.005), shoulder abductors (P=0.002), shoulder flexors (P=0.004), hamstrings (P<0.001), and quadriceps values (P<0.001). In addition, there were improvements in CFQ-R-emotion (P=0.002), treatment burden (P=0.002), vitality (P=0.011), and physical scores (P=0.026), and a reduction in the Borg resting fatigue score (P=0.037). The interleukins levels did not change after the intervention. In adult CF patients with pulmonary exacerbation, early hospital rehabilitation had a significant impact on improving resting fatigue, muscle strength, and quality of life.

Suprathel(®) for severe burns in the elderly: Case report and review of the literature.

Large burns in aged patients are common and treatment often reveals challenging. Cardiovascular complications significantly contribute to the unfavorable prognosis in this group of high-risk patients. Pain medication and sedation can negatively influence cardiovascular stability. Suprathel(®) is well-known for its almost pain free application and reduction of dressing change intervals, and thus lowers the demand for potentially harmful analgesics and sedatives. We present the case of an 81-year-old patient with 51% of total burned body surface area (ABSI=12), who was completely treated with Suprathel(®). Despite a predicted mortality of more than 80%, the patient survived and was discharged home without significant handicaps 69 days after burn. We hypothesize that Suprathel(®) beneficially contributed to the favorable clinical course of this critical patient as less frequent wound-dressing changes did not induce additional pain or sedative medication and thus improved cardiovascular stability.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia.

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

Expression of MUC-1 epitopes on normal bone marrow: implications for the detection of micrometastatic tumor cells.

PURPOSE: The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS: MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS: Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION: Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

Extrathymic T cell differentiation in vitro from human CD34+ stem cells.

Although it is well established that T cells are derived from CD34+ stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in vitro from CD34+ cells in the absence of any thymic influence. We now present a limiting dilution cloning culture system that supports the differentiation of highly purified human CD34+ cells to CD3+ T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum-free medium supplemented with a cocktail of cytokines including flt-3 ligand, interleukin-3 (IL-3), stem cell factor (SCF), and IL-2. CD4+ T cell clones capable of mitogen-stimulated proliferation and response to IL-2, and expressing a varied TCR-Vbeta repertoire were obtained under these conditions. This culture system therefore supports human T lymphopoiesis in the absence of any thymic influence and may prove useful for the evaluation of extrathymic T cell differentiation in vitro.

The monoclonal antibody HCA2 recognises a broadly shared epitope on selected classical as well as several non-classical HLA class I molecules.

HCA2 is a widely used monoclonal antibody, thought to be highly selective for HLA-A and -G heavy chains. We demonstrate here that it also shows affinity to HLA-B73 and HLA-E molecules on intact cells. By comparing the differences in the amino acid (AA) sequences of several HLA class I alleles that are either recognised or not recognised by HCA2, a likely epitope of HCA2 has been deduced. It extends from position 76 to position 83 of the alpha1-domain. In intact cells, the solvent-exposed AA in positions 76 (Ala, Val, or Met), 80 (Asn or Thr) and 83 (Gly) are likely to constitute the recognition region. Inhibition experiments with peptides spanning the region of the alpha1-domain from position 74 to 85 of various HLA class I heavy chains prove that HCA2 recognizes a broadly shared epitope on HLA-E, -F and -G molecules as well as selected HLA-A, -B and -C antigens.