Failed or inadequate bone marrow aspiration: a fast, simple and cost-effective method to produce a cell suspension from a core biopsy specimen.

Failure to aspirate bone marrow (BM) diminishes diagnostic accuracy and efficiency because BM cell suspensions are crucial for modern haematological diagnostic methods such as cytomorphology, flow cytometric immunophenotyping (FCI), cytogenetics or fluorescent in situ hybridization (FISH). We mechanically disaggregated unfixed BM core biopsies with the Dako Medimachine in 65 cases of macroscopically suspected dry taps. Cytospins, three-colour FCI and in some cases karyotyping and FISH were performed successfully. Most cytospins (34 of 50; 68.0%) were of good quality, while a further 18.0% showed moderate but still informative quality. FCI showed good quality in 36 of 60 (60.0%) cases; in 13.3% quality was moderate, but diagnostically useful results were obtained. Surprisingly, all four cases of formerly undiagnosed BM-carcinosis could be clearly detected on cytospins. Finally, five of seven (71.4%) attempts yielded analysable metaphases mostly in cases where no metaphases could be obtained from BM or peripheral blood. The described method of mechanical disaggregation of unfixed BM core biopsies compares favourably with other published approaches, allowing the application of all techniques where BM cell suspensions are needed. Thus, it can help to establish the underlying diagnosis in patients with abnormalities in peripheral blood and unsuccessful marrow aspirations.

Sequence analysis of the mannose-binding lectin (MBL2) gene reveals a high degree of heterozygosity with evidence of selection.

Human mannose-binding protein (MBL) is a component of innate immunity. To capture the common genetic variants of MBL2, we resequenced a 10.0 kb region that includes MBL2 in 102 individuals representing four major US ethnic groups. In all, 87 polymorphic sites were observed, indicating a high level of heterozygosity (total pi=18.3 x 10(-4)). Estimates of linkage disequilibrium across MBL2 indicate that it is divided into two blocks, with a probable recombination hot spot in the 3' end. Three non-synonymous SNPs in exon 1 of the encoding MBL2 gene and three upstream SNPs form common 'secretor haplotypes' that can predict circulating levels. Common variants have been associated with increased susceptibility to infection and autoimmune diseases. The high frequencies of B, C and D alleles in certain populations suggest a possible selective advantage for heterozygosity. There is limited diversity of haplotype structure; the 'secretor haplotypes' lie on a restricted number of extended haplotypes, which could include additional linked SNPs, which might also have possible functional implications. There is evidence for gene conversion in the region between the two blocks, in the last exon. Our data should form the basis for conducting MBL2 candidate gene association studies using a locus-wide approach.

Programmed cell death in normal fetal rat lung development.

Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.

Characterization of an axonemal dynein heavy chain expressed early in airway epithelial ciliogenesis.

The most conspicuous evidence of airway epithelial maturation and vitality is the presence of motile cilia. In an effort to generate genetic and antigenic markers of airway maturation, injury, and repair, we characterized airway epithelial expression of a gene identified by two human expressed sequence tags that encoded peptides with sequence similarity to an invertebrate ciliary dynein heavy chain (DHC). Molecular analyses showed that the gene has a very large RNA transcript that encodes a very high molecular weight polypeptide with biochemical properties that are characteristic of a dynein heavy chain. Expression of the gene transcript correlated with the presence of ciliated cells in tissues, and immunohistochemical localization of the gene product confirmed its presence in the cilia of mature airway epithelium. In epithelium undergoing ciliogenesis ex vivo, expression of the gene transcript preceded ciliation of the epithelium and the gene product was present in the cytoplasm and at the apical border of nonciliated cells. These data suggested that the gene encodes an axonemal DHC that is expressed early during ciliogenesis, before the appearance of cilia.

Regulation of the rat BB1 RNA during normal rat lung development.

BB1 was recently cloned from the WI-38 human fetal lung cell line. Human BB1 (hBB1) is expressed by multiple tissues, including lung. Because inhibition of BB1 translation using antisense oligodeoxynucleotides resulted in prevention of G1 traversal in cultured cells, we hypothesized that BB1 gene expression would be regulated during lung development with greater expression during periods of active lung growth. To gain insight into the expression of BB1 during lung development, a rat BB1 (rBB1) homologue was cloned and used in Northern hybridization analyses and in situ hybridization histochemistry (ISHH). Northern hybridization analyses of fetal and postnatal rat lung demonstrate that rBB1 RNA abundance is relatively low on fetal days E17 through E19, with a small peak of expression occurring on fetal day E20, then increases at birth with peak expression in adult lung. ISHH correlates with the Northern hybridization data and reveals rBB1 RNA expression throughout lung from E17 to E21 in both epithelium and mesenchyme. In postnatal lung, more intense expression of BB1 was observed than in fetal lung, localizing BB1 transcripts to proximal and distal airways and mesenchymal cells surrounding airways. Proliferating cell nuclear antigen (PCNA) was identified in lung sections adjacent to those used for ISHH and it was found that BB1 expression was present in PCNA-positive cells; however, BB1 expression was not limited to PCNA-positive cells in either the fetal or postnatal periods. This was most apparent in adult (60-day) rat lung where essentially no PCNA-positive cells were detected, but intense BB1 expression was detected in airway epithelium and surrounding mesenchyme. These studies demonstrate developmental regulation of BB1 during lung development. The findings are consistent with BB1 action in cell growth-related processes of fetal and early postnatal lung; however, the distribution of BB1 expression in relation to PCNA localization suggests that BB1 participates in cellular functions in addition to cell proliferation.

Successful treatment of obstructive sleep apnea with use of nasal continuous positive airway pressure in three patients with mucosal hemangiomas of the oral cavity.

Cysts and benign tumors are uncommon causes of obstructive sleep apnea (OSA), and surgical removal is usually favored. In patients in whom an operation poses a high risk, however, nasal continuous positive airway pressure (CPAP) may prove beneficial. We describe three patients with hemangiomas of the oral cavity in whom polysomnography revealed moderate to severe OSA. In all three patients, nasal CPAP effectively decreased sleep-related disordered breathing events and dramatically improved their sleep. To our knowledge, this is the first report of OSA associated with hemangiomas involving the upper airway. Our experience suggests that nasal CPAP therapy is effective and well tolerated in such patients.

Effect of fasting on insulin receptor-related receptor messenger ribonucleic acid in rat kidney.

The insulin receptor-related receptor (IRR), a member of the insulin receptor tyrosine kinase family, has structural homology to the insulin receptor (IR) and the IGF-I receptor (IGF-IR). The ligand, gene regulation and biological function of the IRR are not known. Because mRNAs for both the IR and IGF-IR are increased by nutrient restriction, we used RNase protection assays to assess the effects of fasting 48 h on IRR mRNA in kidneys of rats. We compared the changes in IRR with those in IR and IGF-IR mRNAs. We observed a significant increase in steady state levels of IRR (ratio of IRR mRNA to beta-actin in fed P<0.01), suggesting that the ligand for IRR also might be regulated by nutrients.

Long-term clinical experience with transtracheal oxygen catheters.

OBJECTIVE: To evaluate and discuss the use of transtracheal oxygen catheters for the treatment of chronic hypoxemia and to discuss the complications associated with the placement and care of these devices. DESIGN: We conducted a retrospective study at a tertiary medical center and reviewed the pertinent literature. MATERIAL AND METHODS: The medical records of 56 patients who received a transtracheal oxygen catheter between January 1987 and June 1992 at our institution were reviewed for demographic data, diagnosis leading to catheter placement, complications related to catheter use, reason for catheter removal, and duration of use. Follow-up results were established by documentation in the medical records or telephone interview. RESULTS: During the study period, 39 men and 17 women received a transtracheal catheter. More than half the patients (52%) had chronic obstructive pulmonary disease. The duration of use of the catheter ranged from 2 days to more than 6 years, and the most frequent cause for removal of the catheter was death. Of the 56 patients, 42 died with the catheter in place, 24 within the first year after placement. Complications ranged from mucous plugging (38 % of patients) to pneumothorax (4%), and no patient died of a catheter-related complication. Overall, 55% of patients had their catheter for less than 1 year after placement. CONCLUSION: In patients with transtracheal oxygen catheters, problems related to mucous plugging are common, but severe complications such as pneumothorax and pneumomediastinum are uncommon. Although selection factors that would identify ideal candidates for transtracheal oxygen therapy have not been established, such a catheter is best placed in highly motivated patients who can physically manage the daily care of this device.

Two cases of fibrocystic breast disease with polysomy 18 as the sole clonal cytogenetic abnormality.

In the present study, we describe the occurrence of numerical alterations of chromosome 18 in two cases of benign fibrous/fibrocystic tumors of the breast, both of which were studied by conventional cytogenetic investigations and one of which was additionally tested by fluorescence in situ hybridization with the use of an alphoid centromeric probe specific for chromosome 18. Case 1 showed a tetrasomy 18 in 2 of 33 metaphases as the only clonal chromosomal aberration. Case 2 revealed both trisomy and tetrasomy 18 as clonal alterations in metaphases and interphase nuclei.

Expression of the insulin-like growth factor system in postpneumonectomy lung growth.

The insulin-like growth factors (IGF-I and IGF-II) may play an important role in postpneumonectomy compensatory lung growth by translating hormonal inputs and mechanical forces into cellular proliferation signals. We examined the mRNA abundance of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in lungs of rats on postoperative days 1, 2, 3, 5, and 7 following left pneumonectomy (PNX) or shamoperation (SC) and in normal animals (CON). There was no difference in the abundance of lung IGF-I mRNA (measured by Northern analysis) or serum IGF-I (measured by radioimmunoassay (RIA)) between SC and PNX animals. IGF-II mRNA abundance was initially decreased following PNX (73% decrease compared to SC animals on day 1, p < .05) and then rose to approach SC group values on subsequent days. Transcripts for IGFBP-2, -3, -4, -5, and -6 were decreased in both the SC and PNX groups compared to CON animals on the day following pneumonectomy, then rose back to baseline by postoperative day 2-3. Tissue IGFBPs, measured by ligand blot analyses, were not different in either the SC or PNX groups. In contrast, all serum IGFBP bands were increased on postoperative day 1 following either sham or PNX surgery. In addition, serum IGFBP-4 was increased in PNX animals compared to the SC group on days 1 and 2 (increase of 38% and 78%, respectively, p < .05). We conclude that the changes observed in lung IGF and IGFBP expression following pneumonectomy do not represent major.

"Postpolio" sequelae and sleep-related disordered breathing.

OBJECTIVE: To analyze the clinical manifestations and various types of sleep-related disordered breathing (SRDB) in patients with a history of poliomyelitis and with current "postpolio" sequelae (PPS). MATERIAL AND METHOD: We retrospectively reviewed the medical records of 108 consecutive patients with PPS and sleep disturbances encountered during an 11-year period at Mayo Clinic Rochester and abstracted the features of acute polio, PPS, and results of sleep evaluation (overnight oximetry or polysomnography). Only those patients who were not receiving ventilatory support were included in the study. RESULTS: The features of PPS were dyspnea, fatigue, new weakness, and musculoskeletal pain. Of the 108 patients, 35 fulfilled the inclusion criteria. Sleep evaluations revealed three general types of disturbances: obstructive sleep apnea (group O, N = 19); hypoventilation (group H, N = 7); and both (group OH, N = 9). The mean apnea/hypopnea index was 37, 4, and 16 per hour in patients in groups O, H, and OH, respectively (P < 0.05), and the mean arterial carbon dioxide tension was 39, 60, and 55 mm Hg in these respective study groups (P < 0.05). The overall mean age at onset of symptoms of SRDB was 47 years, and the mean latent period after acute polio was 37 years. Hypersomnolence was the commonest SRDB symptom, present in 32 of the 35 patients. Snoring was noted in 100% of patients in group O, 0% in group H, and 67% in group OH. Patients in group O were obese and had normal lung function. Patients in group H tended to have normal weights and a history of diffuse neurologic deficits involving the trunk during the acute episode of polio. Scoliosis, restricted lung function, cor pulmonale, and decreased maximal respiratory pressures were common in patients in group H. Patients in group OH had overlapping features of those in groups O and H. CONCLUSION: In patients with PPS, we identified three patterns of sleep disturbances--obstructive sleep apnea, hypoventilation, and a combination of both. These groups are characterized by clinical features and by results of arterial blood gas determinations, overnight oximetry, and polysomnography. SRDB is a late sequela of poliomyelitis, and clinical evaluation should include information about sleep.

Connexin 26 expression in human and ferret airways and lung during development.

Coordinated microscopic and molecular biological studies were used to document gap junction expression during postnatal development in ferret tracheal epithelium and lung and in fetal and adult human airway and lung. Expression of connexin 26 (Cx26) in the ferret airways was limited to the epithelial layer and was observed only during the newborn interval. In contrast, we found Cx26 expressed in the alveolar epithelium of the ferret lung by in situ hybridization, Northern blotting, RT-PCR amplification, and immunocytochemical labeling at all ages examined. This finding was further confirmed by documentation of gap junctional plaques upon ultrastructural examination of freeze-fracture replicas of adult ferret lung tissue. Parallel studies of developing human fetal lung and airway suggested connexin expression in the airways only in the first trimester but, as in the ferret, persistent expression was observed in both fetal and adult lung. These studies suggest that the transient expression of Cx26 is a reliable early indicator of airway epithelial development and differentiation in the airways. In contrast, Cx26 expression persists throughout life in the lung, suggesting that gap junctions serve more perennial intercellular communication functions in the peripheral lung.

Re-emergence of a fetal pattern of insulin-like growth factor expression during hyperoxic rat lung injury.

Chronic injury to the developing lung results in cell proliferation and characteristic architectural changes. It is likely that growth factors produced and acting locally are important to these processes. Insulin-like growth factors I and II (IGF-I and IGF-II) are peptide growth factors expressed by lung cells. Roles for IGF-I and IGF-II in lung injury are suggested by their expression during lung development and by studies showing changes in IGF-I expression by activated alveolar macrophages, and increases in IGF-II peptide in oxidant arrested alveolar epithelial cells. To investigate whether the expression of IGF-I and IGF-II are changed with hyperoxic exposure, newborn rats were exposed to 80-90% oxygen for up to 6 wk and Northern hybridization analyses, in situ hybridization histochemistry, immunohistochemical staining, and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed. Northern hybridization analyses of RNA extracted from whole lung showed increases in IGF-I and IGF-II mRNAs with prolonged hyperoxia. In situ hybridization histochemistry and immunohistochemical staining demonstrated spatial patterns of IGF-I and IGF-II expression similar to those seen during fetal lung development. In addition, alveolar macrophages express IGF-I and type II epithelial cells express IGF-II in control and oxygen-injured lung. These results suggest that in lung injury resident lung cells may re-express IGFs in a manner reminiscent of fetal development, and activated inflammatory cells may contribute to the proliferative response through autocrine and paracrine mechanisms.

Changes in decorin expression with hyperoxic injury to developing rat lung.

Proteoglycans are extracellular matrix components that appear to play important roles in lung development and in the response to injury. Decorin, a small extracellular matrix-associated proteoglycan, is known to be involved in collagen fibrillogenesis and is a likely participant in the pathogenesis of lung injury. We hypothesized that chronic exposure of the developing lung to hyperoxia would result in temporal and spatial changes in decorin expression. To determine the expression of decorin in normal and oxygen-injured lung, newborn rats were exposed to hyperoxia for 6 wk. Decorin mRNA abundance was determined using Northern hybridization analyses, and decorin expression was localized by in situ hybridization and immunohistochemistry. Decorin mRNA expression in type II pneumocytes was studied using reverse transcription-polymerase chain reaction. Oxygen exposure is associated with a 77% reduction in decorin mRNA in whole lung and a decrease in decorin immunoreactivity in connective tissues surrounding large airways and blood vessels, but an increase in decorin mRNA and protein expression at the tips of alveolar septa. Studies using isolated cells indicate that macrophages and polymorphonuclear neutrophils contain decorin core protein but not decorin mRNA. Type II pneumocytes do not contain either decorin mRNA or core protein. These findings demonstrate that hyperoxic lung injury is associated with localized changes in decorin expression, changes that are not reflected in whole lung RNA studies. It is likely that regional changes in lung decorin expression are influenced by factors produced and acting locally, and that such changes may contribute to the morphologic alterations characteristic of oxygen-induced lung injury.

alpha1-antitrypsin gene mutation hot spot associated with the formation of a retained and degraded null variant [corrected; erratum to be published].

Null alpha1-antitrypsin (alpha1AT) alleles represent the end of a continuum of variants associated with profound alpha1AT deficiency and an increased risk of emphysema. This study characterizes the molecular basis of QOclayton, a new example of an alpha1AT null allele arising from a mutational hot spot in the alpha1AT gene. The QOclayton allele is identical to the normal M1(V213) alpha1AT allele except for an insertion of a cytosine. This insertion occurs in the alpha1AT sequence which normally has seven cytosines corresponding to amino acid residues 360 to 362. The QOclayton mutation is located in the same reiterated DNA sequence as the alpha1AT QObolton deletion mutation and the insertion mutation allele QOsaarbruecken. The QOclayton cytosine insertion causes a 3' frameshift and results in the formation of a termination codon at residue 376, the same consequence as the alpha1AT QOmattawa mutation (L353 T-insertion with a 3' frameshift). To determine the molecular mechanisms responsible for the absence of alpha1AT associated with the QOclayton gene, an in vitro model of QOclayton was established using Chinese hamster ovary cells (CHO) transfected with the QOclayton gene. These cells were evaluated for alpha1AT mRNA expression, protein synthesis and secretion. Although the QOclayton gene expresses a similar amount of alpha1AT mRNA as compared with the normal alpha1AT gene, no QOclayton protein is secreted. Protein trafficking and double-label immunofluorescence demonstrate that the QOclayton protein is retained in the rough endoplasmic reticulum or pre-Golgi compartment and is degraded (t1/2 = 6.5 h). Since QOmattawa, QObolton, and QOsaarbruecken have similar termination sites in the alpha1AT mRNA, they may share a similar intracellular fate.

Significance of clonal chromosome aberrations in breast fibroadenomas.

Despite the high frequency of fibroadenomas of the breast, cytogenetic results are relatively limited. We describe our cytogenetic findings in 30 fibroadenomas. Of these, three showed clonal chromosome abnormalities, i.e., 46,XX,der(6)t(1;6)(q25;p21.3); 48,XX,del(6)(q21),r(11)(?),der(14)t(6;14)(q21;q32),+2mar; and 47,XX,+5.

Fluorescence in situ hybridization studies on breast tumor samples for distinguishing between different subsets of breast cancer.

OBJECTIVES: To evaluate fluorescence in situ hybridization for distinguishing between malignant and benign breast tumors and determining genetic subgroups of breast cancers. STUDY DESIGN: Touch preparations from 94 surgically removed breast tumors (17 benign and 77 malignant) were hybridized with a DNA probe specific for centromeric DNA sequences of chromosome 1. Twenty samples were additionally hybridized with a chromosome 9-specific probe. RESULTS: We investigated the heterogeneity of the cell populations on the basis of the number of signals per nucleus. All benign tumors showed two signals per nucleus. In contrast, carcinomas revealed a broad spectrum of hybridization patterns. Some showed almost exclusively two signals per nucleus, and others exceeded four signals. CONCLUSION: The hybridization patterns of individual tumors can be used for defining different subsets of breast cancer. The results may have prognostic impact, leading to "molecular-cytogenetic grading" of breast cancer.

A fibroadenoma with a t(4;12) (q27;q15) affecting the HMGI-C gene, a member of the high mobility group protein gene family.

An intracanalicular fibroadenoma of the breast showing a clonal chromosomal aberration t(4;12) (q27;q15) as the sole cytogenetic abnormality is described. In order to narrow down the breakpoint region on chromosome 12 on the molecular level we performed fluorescence in situ hybridization (FISH) analysis with a cosmid pool originating from a YAC-contig overspanning part of the region 12q14-15. We were able to narrow down the breakpoint to an approximately 230kb fragment belonging to the HMGI-C gene which maps within an area recently designated as MAR (Multiple Aberration Region). The chromosomal breakpoints of other frequent benign solid tumors, i.e. lipomas, uterine leiomyomas, and pleomorphic adenomas are clustered within the third intron of that gene.

Cellular localization of messenger RNAs for insulin-like growth factors (IGFs), their receptors and binding proteins during fetal rat lung development.

To gain insight into the role of the insulin-like growth factors (IGFs) in regulating lung development, we have used in situ hybridization histochemistry (ISHH) to examine the ontogeny and sites of expression of IGF-I and IGF-II, IGF binding proteins (IGFBP-1 to IGFBP-6), and IGF cell surface receptors in fetal rat lung from 15 to 21 days of gestation. Both IGF-I and IGF-II mRNAs were expressed throughout the developmental period studied with little change in apparent abundance. IGF-I mRNA localized to mesenchymal cells, especially those surrounding airway epithelium, while IGF-II mRNA, which was somewhat more abundant, localized predominantly to epithelia. The type 1 IGF receptor, the receptor that likely mediates the actions of both IGFs, was expressed widely in virtually all cells, whereas the expression of the type 2 IGF receptor, thought to be involved in IGF internalization and degradation, was confined to the mesenchyme and medial layers of intrapulmonary vessels. As with the IGFs, there was little apparent change in the abundance of IGF receptor mRNAs through fetal development, and the type 2 IGF receptor mRNA was more abundant. The expression of IGFBPs changed significantly during lung development. IGFBP-2, -3, -4, and -5 were expressed from day 15 of gestation, but their sites of expression and ontogeny differed. IGFBP-2 mRNA expression was abundant and constant throughout gestation and was confined to proximal and distal airway epithelia. IGFBP-3 and IGFBP-5 also were expressed by proximal airway epithelia, but also exhibited significant expression in interstitial mesenchyme and in mesenchyme surrounding vessels. The abundance of both increased as gestation progressed (IGFBP-5 greater than IGFBP-3). IGFBP-4 mRNA was confined to interstitial mesenchyme and its abundance peaked at days 16 to 19 of gestation. We found no evidence for expression of either IGFBP-1 or IGFBP-6. We conclude that the expression of IGF-I, IGF-II, and the type 1 IGF receptor throughout gestation in the lung supports a role for the IGFs in lung growth and development. The complex pattern of IGFBP expression (differing sites and ontogeny of expression) suggests that the IGFBPs modulate IGF actions at specific target sites. Furthermore, because there is little change in the expression of IGFs or IGF receptor mRNAs during fetal lung development, regulation of IGFBP expression may be essential to the control of IGF actions during lung development.