Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons.

We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor III (ECMR III), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HT1080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonoverlapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O- and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.

Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein.

Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion.

Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species.

The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL-14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino-acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination.

A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.

Technical aspects of the Naval Dosimetry Center quality assurance programme.

The purpose of this paper is to describe the technical aspects of the Naval Dosimetry Center (NDC) quality programme. The Navy has been formally monitoring personnel for occupational exposure to ionising radiation since at least 1946. The current system, the DT-702/PD, is the Harshaw 8840 holder and 8841 card. New card and holder checks are performed to verify that the correct LiF elements and holder filters are in the correct location and are of the correct composition. Element correction coefficient (ECC) magnitude and repeatability are also verified. Several quality assurance parameters are checked by a specially designed shipping machine. Calibration cards are used to calibrate each reader and quality control cards are inserted throughout a group of field cards to verify reader operation during the read process. The success of the programme is measured by annual proficiency tests administered by the National Voluntary Laboratory Accreditation Programme and Pacific Northwest National Laboratories.

The application of LiF:Mg,Cu,P to large scale personnel dosimetry: current status and future directions.

LiF:Mg,Cu,P is starting to replace LiF:Mg,Ti in a variety of personnel dosimetry applications. LiF:Mg,Cu,P has superior characteristics as compared to LiF:Mg,Ti including, higher sensitivity, improved energy response for photons, lack of supralinearity and insignificant fading. The use of LiF:Mg,Cu,P in large scale dosimetry programs is of particular interest due to the extreme sensitivity of this material to the maximum readout temperature, and the variety of different dosimetry aspects and details that must be considered for a successful implementation in routine dosimetry. Here we discuss and explain the various aspects of large scale LiF:Mg,Cu,P based dosimetry programs including the properties of the TL material, new generation of TLD readers, calibration methodologies, a new generation of dose calculation algorithms based on the use of artificial neural networks and the overall uncertainty of the dose measurement. The United States Navy (USN) will be the first US dosimetry processor who will use this new material for routine applications. Until June 2002, the Navy used two types of thermoluminescent materials for personnel dosimetry, CaF2:Mn and LiF:Mg,Ti. A program to upgrade the system and to implement LiF:Mg,Cu,P, started in the mid 1990s and was recently concluded. In 2002, the new system replaced the LiF:Mg,Ti and is scheduled to start replacing the CaF2:Mn system in 2006. A pilot study to determine the dosimetric performance of the new LiF:Mg,Cu,P based dosimetry system was recently completed, and the results show the new system to be as good or better than the current system in all areas tested. As a result, LiF:Mg,Cu,P is scheduled to become the primary personnel dosimeter for the entire US Navy in 2006.

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.

Sequence of the bovine CD44 cDNA: comparison with human and mouse sequences.

CD44 is a cell-surface glycoprotein involved in leukocyte adherence, T-cell activation and lymphocyte homing. We have isolated and sequenced a cDNA clone which encodes for bovine CD44. The predicted amino acid sequence of bovine CD44 has an overall high similarity with that of human and mouse CD44, 79.5 and 73.2%, respectively. In all three species, CD44 has a similar transmembrane region and cytoplasmic tail. In addition, all of the cysteine residues and a majority of the putative N-linked glycosylation sites in the extracytoplasmic domain are conserved between bovine, human and mouse. All three species have an area of low interspecies similarity within the extracytoplasmic domain. This area has a similarity of 34% between bovine and human, 27% between bovine and mouse, and 35% between human and mouse. The location of this area of low similarity is conserved between species.

The erythropoietin receptor gene: cloning and identification of multiple transcripts in an erythroid cell line OCIM1.

The hormone erythropoietin is necessary for the maintenance, generation, and maturation of erythroid cells. This kidney-derived glycoprotein binds to cell-surface receptors, mediating both the proliferation and differentiation of erythroid cells. We have characterized 16 human erythropoietin receptor cDNA clones from two cDNA libraries prepared from OCIM1 poly(A)-containing mRNA. Of these 16 isolates, many showed evidence of incorrect RNA splicing events; others contained small deletions yielding multiple transcripts that may play a role in their erythroleukemic state. The human erythropoietin receptor, as deduced from the sequence of these clones, encodes a 508-amino-acid molecule, including a 24-residue signal peptide, a 226-amino-acid external domain, a transmembrane spanning region of 22 amino acids, and a cytoplasmic domain of 236 amino acids. There is an 80% overall homology between the human and murine erythropoietin receptor sequences.

Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation.

Direct cell-cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell-cell contact in both HOBs and BMCs, colocalized with beta-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a beta-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell-cell adhesion and completely prevented BMP-2-induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.

Phase I study of a humanized anti-CD11/CD18 monoclonal antibody in multiple sclerosis.

OBJECTIVE: To evaluate the safety, pharmacokinetics, pharmacodynamics, and immunogenicity of a humanized anti-CD11/CD18 monoclonal antibody (Hu23F2G) in patients with multiple sclerosis. METHODS: In this phase I uncontrolled dose escalation study, patients (n = 24) with primary or secondary progressive multiple sclerosis received single intravenous infusions of Hu23F2G (0.01 to 4.0 mg/kg). Study parameters included safety, pharmacology, immunogenicity, and brain magnetic resonance imaging (MRI). RESULTS: Hu23F2G had few adverse effects, but 2 cases of urinary tract infection and 2 cases of gingivitis did occur. Transient leukocytes developed in some subjects receiving > or = 1.0 mg/kg. The pharmacokinetic response was nonlinear, with the area under the curve increasing out of proportion to the increase in dose. The mean terminal half-life increased with dose and was 21.9 (SD, 12.8) hours at the 4.0 mg/kg dose. High saturation (> 80%) of CD11/CD18 on circulating leukocytes was achieved with doses > or = 0.2 mg/kg. The duration of high leukocyte saturation was dose-dependent, persisting for more than a week at the 4.0 mg/kg dose. A marked decrease in leukocyte migration in response to cutaneous inflammation was observed. Antibodies against Hu23F2G were not detected. The neurologic examinations were stable except for 1 subject who had worsening weakness associated with an infection. No significant changes were noted on brain MRI scans. CONCLUSIONS: Hu23F2G was tolerated at doses that achieved high degrees of leukocyte CD11/CD118 saturation with in vivo inhibition of leukocyte migration. Because this phase I study was not designed to determine the clinical efficacy of Hu23F2G, further studies are needed.

Lymphocyte HEV adhesion variants differ in the expression of multiple gene sequences.

Lymphocyte adhesion to high endothelial venule cells in lymphoid organs of mice is mediated by several cell-surface glycoproteins, one of which, gp90MEL-14, is detected by the MEL-14 monoclonal antibody (mAb). The MEL-14 mAb was used to select two variants of the EL4 cell line, EL4MEL-14-hi and EL4-MEL-14-lo, that have disparate cell surface expression of this adhesion receptor. A cDNA library constructed from EL4MEL-14-hi mRNA was enriched for sequences present at higher levels in EL4MEL-14-hi cells than EL4MEL-14-lo cells. Quantitative analysis of candidate differential clones by RNA probe protection methods identified five clones whose steady-state mRNA levels were increased in the EL4MEL-14-hi cells. One of these clones, DIFF6, is derived from an RNA whose expression level is higher in several cell lines producing high amounts of MEL-14-reactive gp90, and absent or present at lower levels in several cell lines expressing low levels of this glycoprotein. However, DIFF6 does not encode gp90MEL-14. The nucleotide sequence of this clone predicts a relatively hydrophilic protein characteristic of a cytoplasmic or nuclear protein. Present experiments indicate that expression of gp90MEL-14, a cell-surface-adhesion receptor molecule, may be coregulated with additional cytoplasmic or nuclear factors.

Molecular analysis of the variant alloantigen HLA-B27d (HLA-B*2703) identifies a unique single amino acid substitution.

HLA-B27 is a human major histocompatibility complex class I product defined by its antigenic specificity with conventional alloantisera. Detailed studies using monoclonal antibodies, cytotoxic T lymphocytes (CTL), and isoelectric focusing (IEF) gel electrophoresis demonstrated the heterogeneity in the B27 antigen. We have previously identified a unique variant molecule of B27 designated locally as B27d which is distinguished from other B27 variants by isoelectric point, serologic reactivity, and by a cloned CTL recognition. A gene encoding the B27d variant has been cloned and a complete nucleotide sequence has been determined. Compared to the sequence of the prototype B27a, the B27d has a single base substitution at codon 59 (B27a:TAT--B27d: CAT) in exon 2 responsible for Tyr to His substitution. A His residue at this position in the alpha 1 domain is unique among the known class I sequences and this single amino acid change is apparently sufficient to alter the epitope(s) recognized by antibody and cytotoxic T cell receptor. Previous primary structural analysis of the other five B27 variants has revealed differences of two to four amino acids. The combined structural data on the B27 variants indicate that (1) HLA-B27 represents a family of closely related B locus alleles that share the B27 allospecificity and differ by a limited number (one to four) of amino acid substitutions and (2) point mutation as well as gene conversion might be the mechanism responsible for the allelic variation of B27 antigen family.

Influence of litterbags on growth of fungal vegetative structures.

This study reports a test of the hypothesis that mesh litter bags restrict access of vegetative fungal structures to confined substrate. Unconfined birchwood swab sticks lost more dry weight during a field incubation than confined ones. Samples in fine mesh bags lost more weight than those in coarse mesh bags. Significantly higher numbers of mycelial strands and rhizomorphs were found on unconfined samples. The number of fungal structures was significantly correlated with weight loss of individual samples. It is suggested that the inhibitory effect is most significant in environments where higher fungi are the dominant decomposing organisms.

Improvement in physician's office laboratory practices, 1989-1994.

BACKGROUND: In 1986 and 1989, the Centers for Disease Control and Prevention sponsored institutes on Critical Issues in Health Laboratory Practice. It was noted during the institutes that physician's office laboratories were a rapidly emerging site for clinical laboratory testing, yet no comprehensive data were available regarding the practice of clinical laboratory medicine in physician's office laboratories. As a mechanism to begin addressing this void, the Centers for Disease Control and Prevention added questions on clinical laboratory practice to the National Ambulatory Medical Care Survey, a national probability sample of ambulatory care provided by office-based physicians. Data were collected for survey years 1989, 1991, 1993, and 1994. METHODS: Each survey was conducted among a nationally representative, random sample of office-based physicians who provide ambulatory patient care. Sample physicians were enlisted using both mail and telephone contacts. Clinical laboratory data were obtained via telephone by trained field representatives. Weighted univariate and multivariate analyses were performed on responses from each of the 4 survey years. Analyses were repeated after combining survey responses from years 1989 and 1991 and 1993 and 1994 as representative of physician's office laboratory practices before and after implementation of the Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) final rule in 1992. RESULTS: Quality laboratory practice indicators showed significant increases during the study interval, with implementation of the CLIA '88 final rule in 1992 playing a pivotal role. Relative to 1992, enrollment in proficiency testing programs increased from 32.4% to 52.7% (P<.001), use of daily quality control samples increased from 79.2% to 89.0% (P<.001), and use of daily quality control with written instructions for action following a questionable quality control result (quality control with action step documentation) increased from 62.6% to 77.2% (P<.001). The presence of a medical technologist or technician in the office laboratory was also significantly and independently associated with each of the quality indicators. Although the percentage of physician's offices performing on-site testing decreased from 56% to 45% during the survey interval, overall testing volume appeared unchanged. CONCLUSIONS: The quality of clinical laboratory practice in physician's office laboratories improved during the study interval (1989-1994) as measured by the quality indicators used in the study. The association of this improvement with implementation of the CLIA '88 final rule and the presence of a trained laboratory professional in the testing site indicate the importance of minimum practice standards and professional expertise in ensuring use of quality laboratory practices. Overall test volume appeared to be stable despite a decreased proportion of physician's offices at which on-site testing was performed.

Atypical endocervical glandular cells: accuracy of cytologic diagnosis.

Atypical cells thought to be of endocervical glandular origin often cause diagnostic uncertainty in cervicovaginal smears. For this reason consecutive cases of endocervical glandular atypia diagnosed in smears were correlated with subsequent biopsy diagnoses and then retrospectively reviewed. Smears were originally diagnosed as "mild glandular atypia, probably reactive" or "severe glandular atypia, suggestive of adenocarcinoma in situ" (AIS). Biopsy follow-up was obtained on 34 of 58 patients diagnosed with severe endocervical glandular atypia. Nine patients (26%) had AIS, three with concomitant high-grade squamous intraepithelial lesions (HSIL) and tow with invasive adenocarcinoma. Eighteen patients (53%) had HSIL only. Seven had benign changes. Of 152 patients diagnosed with mild glandular atypia, biopsy follow-up was obtained on 40. One patient had AIS; 14 (35%) had HSIL; one had low-grade SIL (LSIL); and 24 (60%) had benign changes. Blinded review of these smears yielded results similar to those in the biopsy follow-up, that is, the prediction of AIS on smears included most cases of AIS, some invasive adenocarcinomas, a significant number of HSIL, cases and a few benign lesions. A review diagnosis of "reactive glandular cells" proved to be HSIL in 31% of cases and AIS in one case. We conclude that patients with a diagnosis of severe glandular atypia in smears may prove to have AIS or invasive adenocarcinoma, but often have HSIL without concomitant AIS. In addition, although "reactive" glandular atypia in smears usually reflects a benign condition, a significant minority of such patients prove to have HSIL.

Evaluation of the ThinPrep Pap test in clinical practice. A seven-month, 16,314-case experience in northern Vermont.

OBJECTIVE: To compare the cytologic diagnoses and specimen adequacy of the ThinPrep Pap Test with historical data within a distinct patient population to assess test performance and its impact on clinical practice. STUDY DESIGN: A total of 16,314 ThinPrep Pap tests were processed and evaluated at Fletcher Allen Health Care over a seven-month period. A subset of 8,574 tests from a selected provider group (cohort) was compared to the historical conventional cervical cytologic smear data from the cohort population for both cytologic diagnoses and specimen adequacy. The cohort consisted of 12 practice groups, including 60 physicians and providers, utilizing the ThinPrep Pap Test as their primary cervical cancer screening sampling technique. Cytologic diagnoses and specimen adequacy were classified using the Bethesda system. RESULTS: Using a three-tiered diagnostic system similar to the Cytyc clinical trials (within normal limits [WNL], atypical squamous cells of undetermined significance [ASCUS]/atypical glandular cells of undetermined significance [AGUS] and low grade squamous intraepithelial lesion and higher [LSIL]+), the ThinPrep method increased the percentage of cases that could be definitively diagnosed as WNL by 1.71%, lowered the percentage of ambiguous or borderline cases diagnosed as ASCUS/AGUS by 26.59% and increased the percentage of cases diagnostic of LSIL+ by 52.15% in the cohort population. Further subdivision by the Bethesda classification showed that the identification of infectious agents increased 25.51% and the detection of high grade squamous intraepithelial lesion/carcinoma increased 55.14%. Concurrently, cases reported as benign cellular changes (reactive/reparative) decreased 23.1%, and the percentage of cases reported as unsatisfactory/"limited by ..." was reduced 52.71%. Histologic correlation of cases reported as squamous intraepithelial lesion revealed that the percentage of patients with subsequent benign biopsies was reduced by 31.7% utilizing the ThinPrep technique. Further, the percentage of ThinPrep patients with histologically confirmed cervical intraepithelial neoplasia (CIN) 1 and CIN 2/3 increased by 16.3% and 9.3%, respectively. CONCLUSION: Implementation of the ThinPrep Pap Test resulted in statistically significant improvements in both diagnostic yield and specimen adequacy, as seen by others in clinical trials. Comparison of results to historical data within a cohort population reinforced earlier data and lent further support to the claim that the ThinPrep Pap Test is "significantly more effective" than the conventional smear in clinical practice.

Evaluation of the ThinPrep processor for fine needle aspiration specimens.

OBJECTIVE: To test the ThinPrep Processor for fine needle aspiration. STUDY DESIGN: One hundred unfixed, surgically removed specimens were aspirated. One pass was directly smeared, fixed and stained with the Papanicolaou technique. The other pass was rinsed in a proprietary fixative, and a single ThinPrep slide was made. Smears were diagnosed without knowledge of the histologic diagnosis. RESULTS: Cellularity and architectural integrity of cell groups were superior on the conventional slides. Preservation and detail of both epithelial and stromal cells were superior with the ThinPrep Processor. Preservation of background material, such as mucus and colloid, was slightly superior on the ThinPrep slides. Diagnostic sensitivity and specificity for malignancy and unsatisfactory rates were all slightly better on the ThinPrep slides. CONCLUSION: The ThinPrep Processor offers an alternative to direct smears in situations in which expertise in slide preparation is not available.