Sampling methodologies and dosage assessment techniques for submicrometre and ultrafine virus aerosol particles.

AIMS: The aerosolization and collection of submicrometre and ultrafine virus particles were studied with the objective of developing robust and accurate methodologies to study airborne viruses. METHODS AND RESULTS: The collection efficiencies of three sampling devices used to sample airborne biological particles - the All Glass Impinger 30, the SKC BioSampler and a frit bubbler - were evaluated for submicrometre and ultrafine virus particles. Test virus aerosol particles were produced by atomizing suspensions of single-stranded RNA and double-stranded DNA bacteriophages. Size distribution results show that the fraction of viruses present in typical aqueous virus suspensions is extremely low such that the presence of viruses has little effect on the particle size distribution of atomized suspensions. It has been found that none of the tested samplers are adequate in collecting submicrometre and ultrafine virus particles, with collection efficiencies for all samplers below 10% in the 30-100 nm size range. Plaque assays and particle counting measurements showed that all tested samplers have time-varying virus particle collection efficiencies. A method to determine the size distribution function of viable virus containing particles utilizing differential mobility selection was also developed. CONCLUSIONS: A combination of differential mobility analysis and traditional plaque assay techniques can be used to fully characterize airborne viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: The data and methods presented here provide a fundamental basis for future studies of submicrometre and ultrafine airborne virus particles.

An aortic dissection in a young weightlifter with non-Marfan fibrillinopathy.

Aortic dissection (AD) is an uncommon condition that occurs mainly in the older patient population (>40 years). It is rare in younger people and is usually associated with trauma, Marfan's syndrome, or pregnancy. We report a case of a young weightlifter who died from an AD, and upon autopsy, was diagnosed as having non-Marfan's fibrillinopathy. We recommend that AD should be considered in symptomatic patients with any family history of early cardiac deaths, a history suggestive of a connective tissue disorder (that is, multiple joint surgeries) or who practise weightlifting.

Blood usage in an Australian intensive care unit: have we met best practice goals?

The transfusion of blood products, especially red cell concentrates, in critically ill patients is controversial and benefits of red cell concentrate transfusion in these patients have not been clearly demonstrated. We performed a prospective observational study to compare best evidence to actual practice of red cell concentrate and other blood product administration in an intensive care unit (ICU) in a university-associated tertiary hospital. All primary admissions during a 28-day period were included in the study and data collected included transfusion of red cells and blood products, patient demographics and ICU and hospital outcome. One hundred and seventy-five admissions were studied; 44% followed cardiac surgery. Forty-one patients (23%) received red cell concentrates in ICU, with 120 units transfused in 61 separate episodes. Other blood product usage was minimal. One third (20/61) of red cell concentrate transfusion episodes were of a single unit. The mean (+/- SD) pre-transfusion haemoglobin was 7.9 +/- 1.1 g/dl. Despite transfusion, such patients left ICU with a lower haemoglobin concentration compared with untransfused ICU patients (9.5 +/- 1.0 versus 10.5 +/- 2.1 g/dl; P < 0.001). Cardiac surgical patients received similar red cell transfusion to general ICU patients. Univariate analysis showed no significant difference in mortality between patients who did or did not receive red cell concentrate transfusion (P = 0.17). However, red cell concentrate transfusion was associated with a reduced adjusted mortality both in ICU (OR 0.13, 95% CI 0.02-0.73) and in hospital at 28 days (OR 0.10, 95% CI 0.02-0.58). The low red cell concentrate and blood product usage in our ICU were consistent with restrictive transfusion practice and selective red cell concentrate transfusion was associated with reduced mortality.

An orthopedic surgeon's guide to interpreting electromyography.

Electrodiagnostic tests such as electromyography (EMG) and nerve conduction velocity studies are commonly ordered during the evaluation of patients with suspected peripheral nerve compression. Although these tests are invaluable extensions of the physical examination, many physicians are unable to interpret the test results, and so they base their operative decisions on electromyographers' impressions. A systematic approach to EMG interpretation allows surgeons to determine the nature and location of lesions as well as the degree of involvement and the viability of affected skeletal muscles.

Coagulation studies in preoperative neurosurgical patients.

Unselected preoperative coagulation testing is known to have low positive yield. However, no study has specifically evaluated neurosurgical patients. A retrospective study of 1211 patients having neurosurgery over a one-year period was therefore conducted. Preoperative test results (activated partial thromboplastin time [aPTT], prothrombin time [PT] and platelet count) and historical factors indicating a potential bleeding tendency were recorded. Abnormality was defined as a test result outside the normal range for our laboratory. Seventeen per cent of all test results were abnormal. However, if abnormality was redefined as a test result indicating potential bleeding tendency (low platelet count, prolonged aPTT and/or PT), only 7.2% of results were abnormal. Many patients had factors on history indicating a potential bleeding tendency, but only a prolonged aPTT, cranial surgery and the use of anti-hypertensive and anaesthetic drugs preoperatively predicted postoperative bleeding. Prolonged aPTT was predictable on history in most patients. We conclude that routine screening of all preoperative neurosurgical patients in our hospital is unnecessary.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

Progressive articular cartilage loss following radiofrequency treatment of a partial-thickness lesion.

Arthroscopic debridement of partial-thickness articular cartilage lesions is a common orthopaedic procedure. Radiofrequency treatment has rapidly gained clinical acceptance despite the lack of prospective studies involving second-look arthroscopy to determine long-term sequelae. We report a case in which a partial-thickness lesion that had been treated with radiofrequency ablation showed progressive thinning of the articular surface.

Development and reproducibility of a brief food frequency questionnaire for assessing the fat, fiber, and fruit and vegetable intakes of rural adolescents.

OBJECTIVE: To describe the systematic development and reproducibility of a food frequency questionnaire (FFQ) designed to meet the specific research requirements of the Goals for Health cancer prevention intervention program for rural middle school children. DESIGN: A 4-step process was used to develop a brief FFQ for scoring intakes of total fat, fiber, and fruits and vegetables. The resulting questionnaire consisted of 25 food frequency items and 10 supplemental questions. Reproducibility of the questionnaire was determined by comparing responses at the beginning and end of a 4-month interval. SUBJECTS: Study subjects were sixth- and seventh-grade students attending middle schools in rural areas of Virginia and upstate New York. Seventh-grade students participated in the pilot study, and sixth-grade students participated in the reproducibility study. The final version of the FFQ was completed twice by 539 sixth graders. After exclusions for missing and unreliable data, the usable sample size was 415. Boys were somewhat more likely than girls to be excluded for missing data. African-American students comprised 32% of the population. STATISTICAL ANALYSES PERFORMED: Each food frequency item was associated with 3 scores--a fat score, a fiber score, and a combined score for the number of servings of fruits and vegetables. Means and standard deviations were determined for nutrient variables, differences between repeat administrations were tested for significance by paired t test, and Pearson correlation coefficients were calculated for nutrients and for individual food items. RESULTS: Correlation coefficients for nutrient scores were 0.58 for fat, 0.49 for fiber, and 0.51 for fruits and vegetables. For individual food items, correlations ranged from 0.24 to 0.59 (mean=0.41). APPLICATIONS/CONCLUSIONS: Using a systematic approach to developing a study-specific FFQ for rural adolescents is feasible. Further, the reproducibility of the Goals for Health questionnaire was demonstrated for the 3 nutrient scores it was designed to measure. This developmental approach may be readily adapted to other populations, study designs, and nutrients of interest. The validity of the questionnaire remains to be tested.

Ex vivo expansion of megakaryocyte progenitors: effect of various growth factor combinations on CD34+ progenitor cells from bone marrow and G-CSF-mobilized peripheral blood.

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34+ cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1); SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34+ cells yielded significantly higher numbers of CD41+ MK progenitors than BM CD34+ cells with any of the growth factor regimens assayed. PB CD34+ cells (2x10[5]) at day 0 generated 1.2 to 1.3x10(6) CD41+ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34+ cells produced 5x10(5) CD41+ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41+ cells were generated in cultures with combinations 2 and 3 (2.3x10[5] and 4.2x10[4], respectively). The addition of MGDF to cultures that were grown with combination 1 for 5 days increased the number of CD41+ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB-derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34+ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP+ cell expansion ex vivo, suggesting the feasibilty of MK expansion ex vivo in patients with prolonged thrombocytopenia posttransplantation. In summary, these data indicate that GCSF-mobilized CD34+ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34+ BMPCs. CD34+/GP+ MK progenitors may be an appropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

Engraftment and development of human CD34(+)-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cell fractions is crucial for predicting their performance after transplant into high-risk patients following high-dose therapy. We report that human umbilical cord blood cells, 78% to 100% of which express the hematopoietic progenitor cell surface marker CD34, can consistently engraft, develop, and proliferate in the hematopoietic tissues of sublethally irradiated NOD/LtSz-scid/scid mice. Engraftment and development of CD34+ cells is not dependent on human growth factor support. CD34+ cells home to the mouse bone marrow (BM) that becomes the primary site of human hematopoietic development containing myeloid, lymphoid, erythroid, and CD34+ progenitor populations. Myeloid, and in particular lymphoid cells possessing more mature cell surface markers, comprise the human component of mouse spleen and peripheral blood, indicating that development proceeds from primary hematopoietic sites to the periphery. Repopulation of secondary recipients with human cells by BM from primary recipients demonstrates the maintenance of substantial proliferation capacity of the input precursor population. These data suggest that the cells capable of initiating human cell engraftment (SCID-repopulating cells) are contained in the CD34+ cell fraction, and that this mouse model will be useful for assaying the developmental potential of other rare human hematopoietic cell fractions in vivo.

Multilineage engraftment in NOD/LtSz-scid/scid mice from mobilized human CD34+ peripheral blood progenitor cells.

Peripheral blood progenitor cells (PBPCs) are now the most widely used source for hematopoietic support of patients in the autologous transplant setting and are increasingly being used for allogeneic transplantation. A reliable model to characterize the in vivo potential of various PBPC subpopulations could be valuable as a preclinical assay to predict the hematopoietic performance in humans of these populations and of products resulting from their manipulations ex vivo. We have used immunocompromised nonobese diabetic/LtSz-scid/scid (NOD/SCID) mice to engraft human CD34+ PBPCs and to study the repopulation characteristics of this progenitor cell fraction. Following myeloablation, intravenous infusion of CD34+ cells consistently produced engraftment and development in mice. Multilineage development occurred in all mice with CD34+ cells, erythroid precursors, and the most immature populations of myeloid cells and B lymphocytes restricted to the mouse bone marrow (BM). More mature populations of myeloid cells and B lymphocytes were peripheralized to the spleen and blood of the animals. This finding suggests that human hematopoiesis in the mice may recapitulate hematopoietic recovery in humans. The provision of human growth factors was not necessary for either engraftment or development of CD34+ cells. When mice were supplemented with growth factors, engraftment levels were unaffected but development was biased toward myeloid production. These findings indicate that providing nonphysiological levels of human growth factors may obscure or enhance the developmental potential of particular progenitor cell populations in this model. Cells capable of initiating colony formation in vitro were maintained in BM during the engraftment period (up to 17 weeks), suggesting that continuous production of myeloid and erythroid precursors occurred from more primitive hematopoietic cells subsequent to engraftment. In comparing results from this study with previous results, it was found that in this model the engraftment potential of CD34+ umbilical cord blood cells is greater than that described here for CD34+ PBPCs. In summary, this model may provide a reliable assay to predict the hematopoietic potential of progenitor cell populations in humans if a correlation for engraftment of identical cell fractions can be established between the two species.

The history of the galaxies.

Astronomical observations now reach far enough back in time, in enough depth and detail, to reveal the history of galaxies since their formation. The early Universe contained a network of gas clouds that filled much of the space between the young galaxies, where stars were forming at a high rate. Since then, intergalactic space has been swept clean, and galaxies have continued to convert the dwindling supply of gas slow into stars.

Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation.

A large volume culture system was developed for the ex vivo expansion of CD34 positive (+) hematopoietic progenitors, using cell donated by 15 patients receiving high-dose chemotherapy with autologous hematopoietic progenitor cell support (AHPCS). Substantial expansion of myeloid (181-fold) and megakaryocyte (41-fold) progenitors cells was demonstrated, using the conditions that we determined to be optimal: CD34+ progenitors cultured unperturbed for 7 (marrow) or 10 (blood) days in Teflon-coated bags with X-Vivo-10 medium containing 10% autologous plasma, 100 ng/ml, respectively, of recombinant stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The studies demonstrated that (a) CD34 selection was necessary to obtain large, clinically relevant numbers of hematopoietic progenitors, (b) the addition of G-CSF to the baseline regimen of SCF/IL-3/IL-6 significantly enhanced the expansion of myeloid progenitors, (c) the addition of IL-1 to SCF/IL-3/IL-6 did not significantly enhance myeloid progenitor cell expansion, (d) CD34+ G-CSF-mobilized peripheral blood progenitor cells (PBPC) produced higher numbers of myeloid progenitors in culture than CD34+ marrow cells, and (e) long-term tissue culture (LTC) assays demonstrate the preservation of long-term initiating cells in ex vivo culture. The short-term and long-term reconstituting capability of CD34+ PBPC cultured in this system remains to be determined and will be evaluated in a clinical trial where they will be used as the sole source of AHPCS following high-dose therapy.

Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain.

Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.