Testing a Generalizable Machine Learning Workflow for Aquatic Invasive Species on Rainbow Trout (Oncorhynchus mykiss) in Northwest Montana.

Biological invasions are accelerating worldwide, causing major ecological and economic impacts in aquatic ecosystems. The urgent decision-making needs of invasive species managers can be better met by the integration of biodiversity big data with large-domain models and data-driven products. Remotely sensed data products can be combined with existing invasive species occurrence data via machine learning models to provide the proactive spatial risk analysis necessary for implementing coordinated and agile management paradigms across large scales. We present a workflow that generates rapid spatial risk assessments on aquatic invasive species using occurrence data, spatially explicit environmental data, and an ensemble approach to species distribution modeling using five machine learning algorithms. For proof of concept and validation, we tested this workflow using extensive spatial and temporal hybridization and occurrence data from a well-studied, ongoing, and climate-driven species invasion in the upper Flathead River system in northwestern Montana, USA. Rainbow Trout (RBT; Oncorhynchus mykiss), an introduced species in the Flathead River basin, compete and readily hybridize with native Westslope Cutthroat Trout (WCT; O. clarkii lewisii), and the spread of RBT individuals and their alleles has been tracked for decades. We used remotely sensed and other geospatial data as key environmental predictors for projecting resultant habitat suitability to geographic space. The ensemble modeling technique yielded high accuracy predictions relative to 30-fold cross-validated datasets (87% 30-fold cross-validated accuracy score). Both top predictors and model performance relative to these predictors matched current understanding of the drivers of RBT invasion and habitat suitability, indicating that temperature is a major factor influencing the spread of invasive RBT and hybridization with native WCT. The congruence between more time-consuming modeling approaches and our rapid machine-learning approach suggest that this workflow could be applied more broadly to provide data-driven management information for early detection of potential invaders.

Photophysical properties of 2-Phenylindole in poly (vinyl alcohol) film at room temperature. Enhanced phosphorescence anisotropy with direct triplet state excitation.

We report the spectral properties of 2-Phenylindole (2PI) embedded in rigid poly (vinyl alcohol) (PVA) film. The 2PI in PVA film shows relatively strong and structured fluorescence with a maximum at 370 nm and surprisingly strong room temperature phosphorescence with an emission maximum of about 500 nm. The dye is highly immobilized in the polymer matrix, thus presenting high fluorescence anisotropy in an isotropic film of about 0.3 at room temperature. The 2-Phenylindole phosphorescence excited in the usual way through the electronic singlet state excitation (S0 â†’ S1 absorption) results in a very low, near zero anisotropy. We now report that we can directly excite the dye to the triplet state T1 and observe high phosphorescence anisotropy similar to the fluorescence anisotropy. The extinction coefficient for S0 â†’ T1 absorption in the PVA matrix is unusually high- only about 3 orders of magnitude lower than S0 â†’ S1 absorption. We consider this direct excitation to indole's triplet state a very significant finding that may lead to many practical applications. The unusually long-wavelength of excitation around 400 nm, much above typical UV absorption, results in a high phosphorescence anisotropy. This provides a new way to study rotational motion of larger biological objects in the microsecond time scale not accessible through typical fluorescence studies.

Expression of epidermal keratins and filaggrin during human fetal skin development.

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin.

Profilaggrin is a large phosphorylated protein (approximately 400 kDa in humans) that is expressed in the granular cells of epidermis where it forms a major component of keratohyalin. It consists of multiple copies of similar filaggrin units plus amino- and carboxy-terminal domains that differ from filaggrin. Proteolytic processing of profilaggrin during terminal differentiation results in the removal of these domains and generation of monomeric filaggrin units, which associate with keratin intermediate filaments to form macrofibrils in the stratum corneum. The amino-terminal domain contains two calcium-binding motifs similar to the EF-hands found in the S-100 family of calcium-binding proteins. In this report, we expressed the 293-residue amino-terminal pro-domain of human profilaggrin as a polyhistidine fusion protein in Escherichia coli, and characterized calcium binding by a 45Ca++ binding assay and fluorescence emission spectroscopy. Fluorescence measurements indicated that the profilaggrin polypeptide undergoes conformational changes upon the removal of Ca++ with ethylenediamine tetraacetic acid, demonstrating the presence of two calcium-binding sites with affinities for calcium that differ ninefold (1.4 x 10(-4) M and 1.2 x 10(-3) M). We suggest that this functional calcium-binding domain at the amino-terminus of human profilaggrin plays a role in profilaggrin processing and in other calcium-dependent processes during terminal differentiation of the epidermis.

CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.

Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.

Two monoclonal antibodies (AKH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts.

Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.

Skin biopsies and scale samples from nine infants and one fetus affected with harlequin ichthyosis (HI) were obtained from eight families. Epidermal differentiation was examined by morphologic and biochemical criteria and cell culture studies. Two striking abnormalities were identified; first, keratin and filaggrin expression were abnormal and varied between cases, and, second, in all cases lamellar granules were absent or abnormal, and intercellular lamellae within the stratum corneum were absent. Three HI phenotypes were distinguished by variable expression of epidermal structural proteins. Cases were classified by the absence (type 1) or presence (types 2 and 3) of keratins K6 and K16 ("hyperproliferative" keratins) and by the presence of profilaggrin in the interfollicular epidermis (types 1 and 2 only). Profilaggrin is apparently not converted to filaggrin, but it is retained in the scale. The block in profilaggrin processing may be due to an inactive phosphatase. Siblings in two families (presenting with types 1 and 2) showed the same type classification suggesting that expression of the phenotype is consistent within families but differs between families. Cultured HI keratinocytes were normal by phase microscopy, but abnormal by electron microscopy with no lamellar granules and extensive stacking of the upper layers. We conclude that harlequin ichthyosis is a genetically heterogeneous group of disorders with altered lamellar granules, intercellular lipids, and variation in expression and/or processing of structural protein markers of normal epidermal keratinization. Furthermore, the lamellar granule and structural protein defects may be indirectly related via a mechanism involving phosphorylation/dephosphorylation.

Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.

Profilaggrin is a large phosphoprotein that is expressed in the granular cells of epidermis where it is localized in keratohyalin. It consists of multiple copies of single filaggrin units plus N- and C-terminal sequences that differ from filaggrin. Profilaggrin is dephosphorylated and proteolytically processed during terminal differentiation to yield filaggrin, which associates with keratin intermediate filaments to form macrofibrils in the lower layers of the stratum corneum. The N-terminal sequence of human profilaggrin comprises two distinct domains; an acidic A domain of 81 amino acids that binds Ca2+, and a cationic B domain of 212 residues. In this report, we further characterize the N-terminal domain by immunohistochemistry and immunoblot analysis using anti-peptide antibodies raised to the A and B regions. All of these antibodies (n = 4) immunostained keratohyalin in the granular layer of human epidermis and also showed some reaction with the lower stratum corneum. In immunoblot studies, the high molecular weight human profilaggrin reacted with both B domain antibodies whereas it showed a weak and variable reaction with A domain antibodies. In addition to profilaggrin, a cationic 32-kDa protein was detected with all N-terminal antibodies. A similar-sized N-terminal peptide was also produced by in vitro proteolysis of human profilaggrin with endoproteinase 1 (PEP1), a protease involved in processing of mouse profilaggrin, and in cultured rat epidermal keratinocytes transfected with a human profilaggrin cDNA construct. Evidence for at least one additional cleavage within the N-terminal domain is shown by immunoreactivity of smaller (16-20 kDa) acidic and basic proteins with A and B domain antibodies, respectively. These results demonstrate that the N-terminal domain is an integral part of profilaggrin in keratohyalin but is proteolytically cleaved from profilaggrin during the terminal differentiation of keratinocytes to yield a 32-kDa peptide.

Detection of beta-defensins secreted by human oral epithelial cells.

Human beta-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of beta-defensins in vitro and in biological fluid using ProteinChip(R) Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of beta-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.

Reduced human IgG anti-ATGAM antibody formation in renal transplant recipients receiving mycophenolate mofetil.

Exposure to the equine-derived polyclonal antithymocyte preparation, ATGAM, frequently elicits human anti-ATGAM antibody formation. The influence of concomitant immunosuppressants on this antiantibody response has not been established. We therefore evaluated IgG antibody formation to ATGAM in 47 patients receiving ATGAM as part of a prospective, randomized, double-blinded study of mycophenolate mofetil versus azathioprine for maintenance immunosuppression after primary cadaveric renal transplantation. All patients received ATGAM for induction of immunosuppression plus methylprednisolone, prednisone, and cyclosporine. In addition, patients were randomized to receive maintenance immunosuppression consisting of either azathioprine (AZA) 1-2 mg/kg/day, mycophenolate mofetil 2 gm/day (MMF2), or mycophenolate mofetil 3 gm/day (MMF3). Patient sequential sera were independently tested for IgG anti-ATGAM antibody by 2 laboratories, which were blinded to treatment arm assignments, using enzyme-linked immunosorbent assays. Both laboratories found significantly greater anti-ATGAM antibody formation in group AZA compared with groups MMF2 and MMF3: laboratory 1 reported sensitization rates in the 3 groups of 94% (AZA), 50% (MMF2) (P < 0.02 vs. AZA), and 60% (MMF3) (P < 0.05 vs. AZA); and laboratory 2 reported rates of 67% (AZA), 17% (MMF2) (P < 0.02 vs. AZA), and 10% (MMF3) (P < 0.02 vs. AZA). In addition, fewer patients formed high titer antibody in the MMF arms compared to the AZA arm: 56% (AZA), 0% (MMF2) (P < 0.02 vs. AZA), and 20% (MMF3) (P < 0.02 vs. AZA) of patients for laboratory 1; and 20% (AZA), 0% (MMF2) (P < 0.05 vs. AZA), and 0% (MMF3) (P < 0.05 vs. AZA) of patients for laboratory 2. Differences in test results between the 2 laboratories were explained by differences in the sensitivity of their respective immunoassays and in the criteria used for assigning a positive result to test specimens. In this protocol, MMF at 2-3 gm/day was associated with a reduced incidence and titer of IgG anti-ATGAM antibody formation compared with standard azathioprine dosing. Although MMF previously has been reported to inhibit T cell responses that mediate acute cellular rejection, this is the first demonstration that MMF significantly inhibits human B cell responses to antigen in vivo.

Effectiveness of a second course of OKT3 monoclonal anti-T cell antibody for treatment of renal allograft rejection.

A second course of OKT3 monoclonal anti-T cell antibody was given to 21 recipients of kidney transplants. Rejections reversed in 43% of patients in whom 95% of rejections had reversed with their initial OKT3 course. Reversal was highly dependent upon the timing of rejection, anti-OKT3 antibody production, and T cell CD3 modulation. Rejections treated greater than 90 days after transplantation were resistant to OKT3 reversal. High-titer anti-OKT3 antibodies prevented OKT3 reversal of rejection, and effective CD3 (the cell surface target of OKT3) modulation was necessary for successful OKT3 reversal of rejection. Reexposure to OKT3 further stimulated anti-OKT3 antibody production and broadened the specificity of the antibodies produced. OKT3 can effectively and safely be used a second time for treatment of early T cell-mediated renal allograft rejections if high-titer anti-OKT3 antibodies have not been made.

Subscapular abscess due to Haemophilus influenzae type B.

Localized abscess formation is a rare but previously described manifestation of Haemophilus influenzae infection. A majority of the reported cases were caused by nontypeable strains of H influenzae. We report a case of an extensive subscapular abscess due to beta-lactamase-negative H influenzae type B. This, to our knowledge, is the first such case described in English literature.

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations--I. Metabolite formation and irreversible binding to cellular macromolecules.

The metabolism of endogenous estrogens, estradiol and estrone, and the irreversible binding of estrogens to cellular macromolecules have been examined and compared in subcellular microsomal and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol, an NADPH-generating system, and denatured DNA, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 3.26 nmoles/mg protein in 1 hr (S.D. 0.39; 7.9% of total steroid) while binding to DNA was found to be 0.288 nmole/mg DNA/mg protein (S.D. 0.025; 0.39% of total steroid). No significant difference was observed between microsomal preparations from untreated, phenobarbital-treated or 3-methylcholanthrene-treated rats. Irreversible binding to proteins was also demonstrated in the intact hepatocyte cell incubations. After 2-hr incubations of estradiol with hepatocytes, 5.9% (S.D. 1.4%) of the steroid(s) was irreversibly associated with cellular proteins (approximately 1.43 pmoles/mg/min). Analysis of the organic-soluble metabolites demonstrated the presence of the catechol estrogens and their metabolites, 2-hydroxyestradiol, 2-hydroxyestrone, 2-methoxyestradiol, and 2-methoxyestrone. Estrone and estriol were also identified. The aqueous-soluble materials isolated from hepatocyte incubations contained glucuronide, sulfate, and apparent thioether conjugates, as determined by liberation from estrogen metabolites by treatment with beta-glucuronidase, sulfatase, and Raney nickel. Thus, extensive primary and secondary metabolism of estrogens occurs in intact hepatocyte incubations. Furthermore, irreversible binding of estrogens to cellular proteins occurs in these intact cells having demonstrated conjugative pathways of metabolism.

Simulating the effects of climate change on the carbon balance of North American high-latitude forests.

The large magnitude of predicted warming at high latitudes and the potential feedback of ecosystems to atmospheric CO2 concentrations make it important to quantify both warming and its effects on high-latitude carbon balance. We analysed long-term, daily surface meteorological records for 13 sites in Alaska and north-western Canada and an 82-y record of river ice breakup date for the Tanana River in interior Alaska. We found increases in winter and spring temperature extrema for all sites, with the greatest increases in spring minimum temperature, average 0.47 °C per 10 y, and a 0.7-day per 10 y advance in ice breakup on the Tanana River. We used the climate records to drive an ecosystem process model, BIOME_BGC, to simulate the effects of climate change on the carbon and water balances of boreal forest ecosystems. The growing season has lengthened by an average of 2.6 days per 10 y with an advance in average leaf onset date of 1.10 days per 10 y. This advance in the start of the active growing season correlates positively with progressively earlier ice breakup on the Tanana River in interior Alaska. The advance in the start of the growing season resulted in a 20% increase in net primary production for both aspen (Populus tremuloides) and white spruce (Picea glauca) stands. Aspen had a greater mean increase in maintenance respiration than spruce, whereas spruce had a greater mean increase in evapotranspiration. Average decomposition rates also increased for both species. Both net primary production and decomposition are enhanced in our simulations, suggesting that productive forest types may not experience a significant shift in net carbon flux as a result of climate warming.

Red blood cell aggregation versus blood clot formation in ionic and nonionic contrast media.

The effects of ionic and nonionic radiographic contrast media on human blood in plastic syringes were investigated in an in vitro double-blind study. Venous blood, which was drawn into plastic syringes containing one of four (iohexol, iopamidol, diatrizoate sodium meglumine, and ioxaglate sodium meglumine) contrast media, was visually inspected at predetermined time intervals before and after mixing. Aliquots of the mixtures of blood and contrast media also were evaluated microscopically. Irregular red blood cell (RBC) aggregates were observed with the nonionic contrast media. Pronounced RBC morphologic alterations occurred with diatrizoate (ionic), and marked crenation was observed with ioxaglate (ionic). Observed aggregates were freely disaggregated in isotonic saline. Recovered supernatant blood from centrifuged aliquots of the mixtures was evaluated for clot formation. There was no evidence of blood clot formation within 1 hour after blood was introduced into syringes containing either ionic or nonionic contrast media. This time period exceeded the normal clotting time, since blood in syringes without contrast media formed clots within 30 to 45 minutes. Both the nonionic and ionic contrast media prolonged coagulation.

The use of CIE for the detection of Clostridium difficile toxin in stool filtrates: laboratory and clinical correlation.

Two CIE procedures (CIE-1, CIE-2) for the detection of Clostridium difficile in diarrheal stools were evaluated by comparison to cytotoxin assay and culture results and by comparison to a clinical likelihood of C. difficile-induced diarrhea. Using a combination of toxin assay and culture results for reference, the CIE-1 and CIE-2 procedures had sensitivities of 33% and 47%, specificities of 89% and 91%, and positive predictive values of 42% and 54%, respectively. Using clinical likelihood for reference, the best results were obtained by the CIE-2 method, which yielded a positive predictive value of 77%. Neither CIE procedure provided an acceptable sensitivity for the detection of C. difficile in stools.

Penicillin susceptibility of Streptococcus pneumoniae in 1978. Screening for resistance by disk testing.

Recently, strains of Streptococcus pneumoniae with greatly increased resistance to penicillin (minimal inhibitory concentrations or MICs, 1--8 microgram/ml) were recovered in cultures of blood from patients in South Africa and from one in Minnesota who had serious pneumococcal infections. The authors undertook a study to determine whether these resistant strains have become prevalent in their locale. Between January and July 1978, the laboratories of hospitals serving the greater Madison, Wisconsin, area (population 200,000) contributed 243 pneumococcal isolates for susceptibility testing by an agar dilution technic. Strains with greatly increased resistance (MICs greater than or equal to 1.0 microgram/ml) were not found; only six strains (2.4%) had relative resistance to penicillin (MICs .125--.50 microgram/ml), a range of susceptibility that has been associated with inconsistent clinical responses to treatment with penicillin. Overall, the susceptibility patterns of these 243 isolates are similar to those reported from other centers in North America over the past 30 years. Routine susceptibility testing of pneumococci by hospital laboratories in our area does not appear to be necessary now, but laboratories are advised to screen blood and spinal fluid isolates by a disk-diffusion method. Studies of these 243 isolates and seven South African multiply-resistant strains using a modified Kirby-Bauer technic, showed that a zone of inhibition less than 35 mm around a 10-unit penicillin disk, or better, less than 17 mm around a 1-microgram oxacillin disk, correlates strongly (P less than .001) with resistance to penicillin (MIC greater than or equal to .1 microgram/ml).

The role of a bioengineered artificial kidney in renal failure.

Renal failure continues to carry substantial burden of morbidity and mortality in both acute and chronic forms, despite advances in transplantation and dialysis. There is evidence to suggest that the kidney has metabolic, endocrine, and immune effects transcending its filtration functions, even beyond secretion of renin and erythropoietin. Our laboratory has developed experience in the tissue culture of renal parenchymal cells, and has now been able to demonstrate the metabolic activity of these cells in an extracorporeal circuit recapitulating glomerulotubular anatomy. We have observed active transport of sodium, glucose, and glutathione. We describe the design and initial preclinical testing of the bioartificial kidney, as well as future directions of our research.

The OKT3 Antibody Response Study: a multicentre study of human anti-mouse antibody (HAMA) production following OKT3 use in solid organ transplantation.

Human anti-murine antibody titres following patient exposure to the monoclonal antibody Orthoclone OKT3 (muromonab-CD3) are determined by laboratories using diverse analytical methods which are not standardized and whose concordance is not established. A multicentre study group therefore compared testing for IgG anti-OKT3 antibody among seven laboratories. A set of 270 sera was obtained from 30 heart, 30 kidney and 30 liver transplant recipients with no previous exposure to OKT3 who were receiving OKT3 for induction immunosuppression. Sera were collected from each patient prior to and at 24 +/- 2 days and 31 +/- 2 days following initial OKT3 exposure. Identical aliquots of all 270 sera were tested for IgG anti-OKT3 antibody by each laboratory. In addition, the limit of detection of each laboratory's method was estimated by titration of an affinity-purified IgG anti-OKT3 reference material of known concentration. Anti-OKT3 antibody formation differed greatly among the three organ groups. Cardiac patients demonstrated the least sensitization and almost exclusively lower titres, while kidney recipients had more frequent and higher titre antibody formation. Liver recipients yielded the highest sensitization rate and the most frequent high titre sera. Importantly, the seven laboratories differed widely in the number of pretreatment sera reported as positive (ranging from 0% to 41% among laboratories), the number of post-OKT3 sera reported as positive (17-63%), the number of post-OKT3 samples with titre > or = 1000 (2-31%), and the number of patients sensitized 19-69%). Concordance among laboratories was highly variable, with interlaboratory agreement ranging from 38% to 83% on the sample titres assigned to 180 post-OKT3 sera. Many of the discordant results were consistent with differences in the limit of detection of the analytical methods, which ranged from 0.19 microgram/ml to > or = 15 micrograms/ml, a nearly 100-fold difference among laboratories. This study demonstrated the presence of both good concordance and significant discordance among laboratories in determining human anti-mouse antibody titres, and demonstrated that common titre categories (100, 1000, 10,000) were not equivalent among laboratories. The level of concordance among methods should be considered when comparing anti-OKT3 antibody results from different centres and their correlation with clinical events. Universal comparative testing, patterned after proficiency testing programmes, is needed to assess differences among laboratories and to bring uniformity and a sound interpretative basis to this field of testing.

Evaluation of C. diff.-CUBE test for detection of Clostridium difficile-associated diarrhea.

The toxin B assay was used to evaluate C. diff.-CUBE, a new dot-immunobinding assay (DIA) for the laboratory diagnosis of Clostridium difficile-associated diarrhea. The widely used latex agglutination test was also included for comparison. Stools from 100 patients suspected of having C. difficile-associated diarrhea were tested. The toxin B assay, latex agglutination, and DIA tests were positive for 12%, 9%, and 22% of the specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of the DIA test were 67%, 84%, 36%, and 95%, respectively, compared with the toxin B assay. The specificity (98%) and positive predictive value (78%) for the latex agglutination test were significantly higher than those of the DIA test. Of 13 specimens solely positive by the DIA test, 11 were cultured and none were positive. Clinical assessment supported only two of the 13 positive DIA results. When clinical assessment was included in the analysis, the DIA positive predictive value rose to 45%. Although the sensitivity and negative predictive values of the DIA test are comparable to the latex agglutination test, the low specificity and positive predictive values of the DIA test make it an inappropriate method to use for screening in a population with a low prevalence of true positives.