Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands functionally compromised post IR.

Comparative analysis of FoxP3(+) regulatory T cells in the target tissues and blood in chronic graft versus host disease.

Activation and migration of regulatory T cells (Treg) into tissue is critical in control of inflammation, but has not been examined extensively in chronic graft versus host disease (cGVHD). In parallel studies of tissues and blood, we determined that FoxP3(+) T cells increased in proportion to T effectors (Teff) in tissue infiltrates in oral and cutaneous lichenoid cGVHD. These FoxP3(+) cells expressed distinguishing phenotypic and functional markers of Treg (CD3(+), CD4(+), CD27(+), ICOS(+) and CD39(+)), not found on FoxP3(-) Teff. Both Teff and FoxP3(+) Treg expressed T-bet and the chemokine receptor CXCR3, however, consistent with a common mechanism of chemokine-mediated migration into tissue. Furthermore, functional markers (ICOS and CD39) and chemokine receptors (CXCR3) were both present in a higher proportion of FoxP3(+) cells in tissues than in peripheral blood, consistent with recruitment and activation of Treg in cGVHD target tissues. Finally, the 'activated' CD45RA(-)FoxP3(hi) subset of Treg cells, which highly express functional markers, were found in comparable frequencies in cGVHD patients and normal controls, despite a significant deficit in naive 'resting' Treg. These findings are consistent with Treg capacity to upregulate functional markers and traffick into tissue in cGVHD.

Bovine AAV transcytosis inhibition by tannic acid results in functional expression of CFTR in vitro and altered biodistribution in vivo.

Bovine adeno-associated virus (BAAV) can enter a cell either through a transcytosis or transduction pathway. We previously demonstrated that particles entering via the transcytosis pathway can be redirected to transduce the cell by blocking particle exocytosis with tannic acid (TA). To investigate whether this approach is useful in lung gene therapy applications, we tested the effect of TA on BAAV transduction in cystic fibrosis airway epithelia in vitro, and in mouse lung in vivo. Our findings suggest that BAAV transcytosis can occur in vivo and that treatment with TA reduces transcytosis and increases lung transduction. TA treatment did not impair the sorting and the activity of the BAAV expressed cystic fibrosis transmembrane regulator membrane protein.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

Immunohistochemical localization of mu-opioid receptors in human dental pulp.

Studies in both animal and clinical models suggest that opioids exert their analgesic effects not only through activation of receptors in the CNS but also through interaction with peripheral opioid receptors. This study evaluated the presence and distribution of mu-opioid receptors in human dental pulp. Human third molars indicated for extraction were removed, fixed in 4% paraformaldehyde and 0.2% picric acid, and decalcified in 10% EDTA and 7.5% polyvinylpyrrolidone. The teeth were cut using a cryostat, and the avidin-biotin peroxidase immunohistochemistry technique was used. Immunostaining for mu-opioid receptors was detected along the nerve bundle of the radicular as well as coronal dental pulp. Positive immunostaining was also observed in the individual nerve fibers in the coronal region. This demonstration of opiate receptors on pulpal nerve fibers suggests a peripheral site in the dental pulp where endogenous or exogenous opioids can interact with mu-opioid receptors.

Tissue compatibility of two biodegradable tubular scaffolds implanted adjacent to skin or buccal mucosa in mice.

Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.

An alternative interpretation of nanobacteria-induced biomineralization.

The reported isolation of nanobacteria from human kidney stones raises the intriguing possibility that these microorganisms are etiological agents of pathological extraskeletal calcification [Kajander, E. O. & Ciftçioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274-8279]. Nanobacteria were previously isolated from FBS after prolonged incubation in DMEM. These bacteria initiated biomineralization of the culture medium and were identified in calcified particles and biofilms by nucleic acid stains, 16S rDNA sequencing, electron microscopy, and the demonstration of a transferable biomineralization activity. We have now identified putative nanobacteria, not only from FBS, but also from human saliva and dental plaque after the incubation of 0.45-microm membrane-filtered samples in DMEM. Although biomineralization in our "cultures" was transferable to fresh DMEM, molecular examination of decalcified biofilms failed to detect nucleic acid or protein that would be expected from growth of a living entity. In addition, biomineralization was not inhibited by sodium azide. Furthermore, the 16S rDNA sequences previously ascribed to Nanobacterium sanguineum and Nanobacterium sp. were found to be indistinguishable from those of an environmental microorganism, Phyllobacterium mysinacearum, that has been previously detected as a contaminant in PCR. Thus, these data do not provide plausible support for the existence of a previously undiscovered bacterial genus. Instead, we provide evidence that biomineralization previously attributed to nanobacteria may be initiated by nonliving macromolecules and transferred on "subculture" by self-propagating microcrystalline apatite.

Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells.

Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.

Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.

Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization.

Human saliva contains histidine-rich proteins, histatins, which have antifungal activity in vitro. The mechanism by which histatins are able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the spheroplasts was saturable and could be competed with unlabeled histatin 3. A single histatin 3 binding site with a K(d) = 5.1 microM was detected. Histatin 3 binding resulted in potassium and magnesium efflux, predominantly within the first 30 min of incubation. Studies with fluorescent histatin 3 demonstrate that the protein is internalized by C. albicans and that translocation of histatin inside the cell is closely associated with cell death. Histatin binding, internalization, and cell death are accelerated in low-ionic-strength conditions. Indeed, a low extracellular salt concentration was essential for cell death to occur, even when histatin 3 was already bound to the cell. The interaction of histatin 3 with C. albicans, and subsequent cell death, is inhibited at low temperature. These results demonstrate that the candidacidal activity of histatin 3 is not due exclusively to binding at the cell surface but also involves subsequent interactions with the cell.

Extracellular matrix protein-induced changes in human salivary epithelial cell organization and proliferation on a model biological substratum.

We have used a denuded rat tracheal preparation as a biological substratum on which to examine the growth and morphology of a salivary epithelial cell line (HSG) in vitro. In the absence of an additional coating of matrix proteins, HSG cells grew at low density on tracheae. Coating the tracheae with Vitrogen (a commercial collagen I preparation) or fibronectin promoted HSG cell growth and monolayer formation. Conversely, if a coating of Matrigel was applied, cells grew in a more organized fashion, but at low density. Generally similar results were obtained with cells grown on laminin and collagen IV but with less organization. These studies demonstrate the utility of a natural, tubular substratum for testing the influence of different matrix proteins on salivary epithelial cell behavior.

Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein.

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+ channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1beta cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1beta cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of approximately 92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

Differences in the metabolism of postprandial lipoproteins after a high-monounsaturated-fat versus a high-carbohydrate diet in patients with type 1 diabetes mellitus.

There is little information comparing the effects of a high-monounsaturated (Mono)-fat versus a high-carbohydrate (CHO) diet in patients with type 1 diabetes mellitus. In the present study, the effects of these diets on a number of metabolic parameters were compared. Seventeen normolipidemic, nonobese patients with type 1 diabetes were provided with the diets for 4 weeks each in a randomized, crossover design. The percentages of Mono fat of the two diets were 25 Mono versus 9 CHO, with a corresponding total fat content of 40% versus 24% and a total CHO content of 45% versus 61%. At the end of each dietary period, parameters of glycemic control, coagulation factors, and fasting and postprandial lipoproteins were assessed. There were no differences in weight, glycemia, insulin dose, fasting lipid profile, or coagulation factors between the two diets. However, the metabolism of postprandial lipoproteins after a fat load differed; viz, after the Mono diet compared with the CHO diet, mean plasma triglyceride levels over 10 hours were higher (P=.0025, by repeated-measures ANOVA). The levels of triglyceride (P=.0045) and retinyl esters (P=.0046) in chylomicrons (Sf>400) and chylomicron remnants (Sf 100 to 400) (P=.0047 and P=.043, respectively), and the total particle number (apolipoprotein B levels) in chylomicron remnants (P=.001) and small, very low density lipoprotein (Sf 20 to 100, P=.016) were also higher. Our data suggest that in patients with type 1 diabetes, a CHO diet might be preferable to a Mono diet, since adherence to the former results in a lower number of circulating postprandial lipoprotein particles that are potentially atherogenic.

Diagnosis and outcome of 100 consecutive patients with extreme granulocytic leukocytosis.

PURPOSE: To determine the clinical features, causes, and prognostic significance of extreme leukocytosis in adults. PATIENTS AND METHODS: Medical records of 100 consecutive patients who presented at the Minneapolis Veterans Affairs Medical Center between March 1993 and January 1994 with more than 25,000 leukocytes/microL blood and with more than 50% granulocytes were reviewed. Demographic, clinical, and outcome information was recorded, and a cause of extreme leukocytosis was sought in each case. RESULTS: Extreme leukocytosis was attributed to infection in 48 cases, advanced malignancy in 13 cases, hemorrhage in 9 cases, glucocorticoids in 8 cases, and other causes in 22 cases. Four patients had previously diagnosed conditions resulting in chronic leukocytosis. Higher leukocyte counts were associated with malignancy (chi2 for trend=12.5, P <0.002). Fever was more common in patients with infection (weighted rate ratio=3.7, 95% Confidence interval [CI]=2.2 to 6.2). Mortality was high overall (31%), and was greater in patients with noninfectious diagnoses compared with infected patients, an association which persisted after stratification by leukocyte count (weighted rate ratio=2.5, 95% CI=1.2 to 4.9). CONCLUSION: Clinicians should be aware that extreme leukocytosis with a predominance of granulocytes is associated with infection in only 48% of cases. The presence of fever increases the likelihood that infection is the cause. Mortality is high, particularly in patients without infection.

Aggregation of the high affinity IgE receptor results in the tyrosine phosphorylation of the surface adhesion protein PECAM-1 (CD31).

One of the earliest events after aggregation of the high affinity receptor for IgE (FcepsilonRI) on mast cells is the activation of protein tyrosine kinases resulting in tyrosine phosphorylation of numerous proteins. Using a monoclonal antibody raised against the rat basophilic leukemia RBL-2H3 cells, we identified that platelet/endothelial cell adhesion molecule 1 (PECAM-1 or CD31) was tyrosine phosphorylated in these cells. Aggregation of PECAM-1 did not induce a detectable increase in its tyrosine phosphorylation, nor did it result in degranulation. However, the minimal tyrosine phosphorylation of PECAM-1 in nonstimulated cells was dramatically increased after FcepsilonRI aggregation. This receptor-induced tyrosine phosphorylation of PECAM-1 was an early event, independent of Ca2+ influx or of the activation of protein kinase C and of cell adhesion. PECAM-1 is an adhesion molecule that is required for the transmigration of leukocytes across the endothelium into sites of inflammation. Therefore tyrosine phosphorylation of PECAM-1 may modulate its interaction with other molecules, thereby regulating the migration of basophils into inflammatory sites.

alpha-Galactosidase A deficient mice: a model of Fabry disease.

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

Anti-ganglioside monoclonal antibody AA4 selectively inhibits IgE-induced signal transduction pathways in rat basophilic leukemia cells.

In rat basophilic leukemia 2H3 (RBL-2H3) cells, mAb AA4 binds to two derivatives of ganglioside GD1b that associate with the Src family kinase p53/56lyn and a serine kinase. Pre-incubation of cells with mAb AA4 blocks immunoglobulin E (IgE) mediated histamine release. In the present study we investigated the effect of incubation with mAb AA4 on signal transduction events. In addition to stimulation of the high affinity IgE receptor (Fc epsilonRI), cells were also activated by the calcium ionophore A23187 and the acetylcholine agonist carbachol in RBL-2H3 cells transfected with the G protein-coupled m3 muscarinic receptor. Incubation of cells with mAb AA4 in a dose-dependent manner inhibited the following Fc epsilonRI-induced signal transduction events: the increase of intracellular free calcium, phosphoinositol breakdown, tyrosine phosphorylation of proteins including the beta- of Fc epsilonRI and secretion. However, there was no inhibition of degranulation or of these biochemical events when cells were stimulated with calcium ionophore or activated via a G protein-coupled pathway. Our results demonstrate that mAb AA4 selectively blocks Fc epsilonRI-induced cell activation at a very early step upstream of receptor tyrosine phosphorylation. As mAb AA4 has previously been found to bind to gangliosides associated with Fc epsilonRI, inhibition of very early biochemical events may be due to interaction of mAb AA4 with the Fc epsilonRI induced signal transduction cascade at the receptor level.

Immunomagnetic isolation of rat bone marrow-derived and peritoneal mast cells.

Mast cells are difficult to purify from heterogeneous cell populations and to preserve, especially for pre-embedding immunostaining at the ultrastructural level. We have developed a technique that permits the isolation of a pure population of mast cells suitable for immunocytochemical studies. A rat mast cell-specific monoclonal antibody (MAb AA4) conjugated to tosylactivated Dynabeads 450 was used to immunomagnetically separate mast cells from rat bone marrow and peritoneal cell suspensions. Approximately 85% of the mast cells were recovered in the positive population that comprised virtually pure mast cells. After microwave fixation, morphological examination showed that the cells were intact and retained their ultrastructural detail. Mast cells in all stages of maturation were immunolabeled with a panel of antibodies after immunomagnetic separation. The combination of immunomagnetic separation followed by immunostaining should prove useful for the study of mast cell maturation and for the characterization of other specific cell types that are present in tissues in only limited numbers.

Diurnal change of blood count analytes in normal subjects.

Short-term, within one 24-hour day (diurnal period) within-person changes of the principal blood count analytes in healthy subjects were studied at three major institutions. The results from each test site were indistinguishable and were therefore combined to make a database of 96 healthy subjects. Analytical imprecision of each analyte was subtracted from the total observed variation to give true diurnal change. Each analyte showed characteristic changes. As would be expected, cellular properties of erythrocytes, such as MCV (mean cell volume) and MCH (mean cell hemoglobin) showed negligible change. The red cell count, hematocrit, and hemoglobin showed changes that were consistent with fluid balance change. Total white cell count and some differential count components showed major changes that raised questions of the confidence limits of clinical decision levels and the validity of commonly used reference intervals. Platelet count changes were typically less than analytic imprecision, suggesting the need for improvement in this aspect of analyzer performance.