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Long-term preservation of laboratory strains of Chlamydomonas reinhardtii has historically involved either liquid nitrogen cryopreservation, which is expensive and labor intensive, or storage on agar plates, which requires frequent transfer to new plates, and which may leave samples susceptible to contamination as well as genetic drift and/or selection. The emergence of C. reinhardtii as a model organism for genetic analysis and experimental evolution has produced an increasing demand for an efficient method to cryopreserve C. reinhardtii populations. The GeneArt™ Cryopreservation Kit for Algae provides the first method for algal storage at -80°C; however, little is known about how this method affects recovery of different clones, much less polyclonal populations. Here, we compare postfreeze viability of clonal and genetically mixed samples frozen at -80°C using GeneArt™ or cryopreserved using liquid nitrogen. We find that the GeneArt™ protocol yields similar percent recoveries for some but not all clonal cultures, when compared to archiving via liquid N2. We also find that relative frequency of different strains recovered from genetically mixed populations can be significantly altered by cryopreservation. Thus, while cryopreservation using GeneArt™ is an effective means for archiving certain clonal populations, it is not universally so. Strain-specific differences in freeze-thaw tolerance complicate the storage of different clones, and may also bias the recovery of different genotypes from polyclonal populations., (© The Author(s) 2021. Published by Oxford University Press.)

Evolutionary transitions in individuality (ETIs) occur when formerly autonomous organisms evolve to become parts of a new, 'higher-level' organism. One of the first major hurdles that must be overcome during an ETI is the emergence of Darwinian evolvability in the higher-level entity (e.g. a multicellular group), and the loss of Darwinian autonomy in the lower-level units (e.g. individual cells). Here, we examine how simple higher-level life cycles are a key innovation during an ETI, allowing this transfer of fitness to occur 'for free'. Specifically, we show how novel life cycles can arise and lead to the origin of higher-level individuals by (i) mitigating conflicts between levels of selection, (ii) engendering the expression of heritable higher-level traits and (iii) allowing selection to efficiently act on these emergent higher-level traits. Further, we compute how canonical early life cycles vary in their ability to fix beneficial mutations via mathematical modelling. Life cycles that lack a persistent lower-level stage and develop clonally are far more likely to fix 'ratcheting' mutations that limit evolutionary reversion to the pre-ETI state. By stabilizing the fragile first steps of an evolutionary transition in individuality, nascent higher-level life cycles may play a crucial role in the origin of complex life.This article is part of the themed issue 'Process and pattern in innovations from cells to societies'., (© 2017 The Author(s).)

In the absence of aqueous buffer, most enzymes retain little or no activity; however, "water-free" enzymes would have many diverse applications. Here, we describe the chemically precise immobilization of an enzyme on an engineered surface designed to support catalytic activity in air at ambient humidity. Covalent immobilization of haloalkane dehalogenase on a surface support displaying poly(sorbitol methacrylate) chains resulted in ∼40-fold increase in activity over lyophilized enzyme powders for the gas-phase dehalogenation of 1-bromopropane. The activity of the immobilized enzyme in air approaches 25% of the activity obtained in buffer for the immobilized enzyme. Poly(sorbitol methacrylate) appears to enhance activity by replacing protein-water interactions, thereby preserving the protein structure.

We report a general and facile method that permits the transfer (stacking) of multiple independently fabricated and nanoscopically thin polymeric films, each containing a distinct bioactive agent, onto soft biomedically relevant surfaces (e.g., collagen-based wound dressings). By using polyelectrolyte multilayer films (PEMs) formed from poly(allyl amine hydrochloride) and poly(acrylic acid) as representative polymeric nanofilms and micrometer-thick water-soluble poly(vinyl alcohol) sacrificial films to stack the PEMs, we demonstrate that it is possible to create stacked polymeric constructs containing multiple bioactive agents (e.g., antimicrobial and antibiofilm agents) on soft and chemically complex surfaces onto which PEMs cannot be routinely transferred by stamping. We illustrate the characteristics and merits of the approach by fabricating stacks of Ga 3+ (antibiofilm agent)- and Ag + (antimicrobial agent)-loaded PEMs as prototypical examples of agent-containing PEMs and demonstrate that the stacked PEMs incorporate precise loadings of the agents and provide flexibility in terms of tuning release rates. Specifically, we show that simultaneous release of Ga 3+ and Ag + from the stacked PEMs on collagen-based wound dressings can lead to synergistic effects on bacteria, killing and dispersing biofilms formed by Pseudomonas aeruginosa (two strains: ATCC 27853 and MPAO1) at sufficiently low loadings of agents such that cytotoxic effects on mammalian cells are avoided. The approach is general (a wide range of bioactive agents other than Ga 3+ and Ag + can be incorporated into PEMs), and the modular nature of the approach potentially allows end-user functionalization of soft biological surfaces for programmed release of multiple bioactive agents.

The persistence of bacterial biofilms in chronic wounds delays wound healing. Although Ga(3+) can inhibit or kill biofilms, precipitation as Ga(OH)3 has prevented its use as a topical wound treatment. The design of a microfilm construct comprising a polyelectrolyte film that releases noncytotoxic concentrations of Ga(3+) over 20 d and a dissolvable micrometer-thick film of polyvinylalcohol that enables facile transfer onto biomedically important surfaces is reported. By using infrared spectroscopy, it is shown that the density of free carboxylate/carboxylic acid and amine groups within the polyelectrolyte film regulates the capacity of the construct to be loaded with Ga(3+) and that the density of covalent cross-links introduced into the polyelectrolyte film (amide-bonds) controls the release rate of Ga(3+) . Following transfer onto the wound-contact surface of a biologic wound dressing, an optimized construct is demonstrated to release ≈0.7 μg cm(-2) d(-1) of Ga(3+) over 3 weeks, thus continuously replacing Ga(3+) lost to precipitation. The optimized construct inhibits formation of P. aeruginosa (two strains; ATCC 27853 and PA01) biofilms for up to 4 d and causes pre-existing biofilms to disperse. Overall, this study provides designs of polymeric constructs that permit facile modification of the wound-contacting surfaces of dressings and biomaterials to manage biofilms., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Silver is a widely used antimicrobial agent, yet, when impregnated in macroscopic dressings, it stains wounds, can lead to tissue toxicity, and can inhibit healing. Recently, polymeric nanofilms containing silver nanoparticles were reported to exhibit antimicrobial activity at loadings and release rates of silver that are 100× lower than conventional dressings. Here, fabrication of composite microfilm constructs that provide a facile way to transfer the silver-loaded polymeric nanofilms onto wounds in vivo is reported. The construct is fabricated from a silver nanoparticle-loaded polymeric nanofilm that is laminated with a micrometer-thick-soluble film of polyvinylalcohol (PVA). When placed on a moist wound, the PVA dissolves, leaving the silver-loaded nanofilm immobilized on the wound-bed. In vitro, the immobilized nanofilms release <1 μg cm(-2) d(-1) of silver over 30 d from skin dermis and they kill 5 log10 CFUs of Staphylococcus aureus in 24 h. In mice, wounds inoculated with 10(5) CFU S. aureus presented up to 3 log10 less bacterial burden when treated with silver/nanofilms for 3 d, as compared to unmodified wounds. In uncontaminated wounds, silver/nanofilms allow normal and complete wound closure by re-epithelialization. Dissolvable microfilm constructs may overcome key limitations associated with current uses of silver in wound healing., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To examine the feasibility and efficacy of a staff-delivered motivational interviewing technique on treatment, diet, medication, and fluid adherence in adult patients receiving outpatient hemodialysis., Design: A pre-post design was used for this pilot study., Sample/setting: Twenty-nine participants were recruited from a non-profit, free-standing hemodialysis clinic in the central United States., Methods: Prior to and after the three-month motivational interviewing intervention, three months of adherence data and the Health Care Climate Questionnaire (HCCQ) were collected., Results: Motivational interviewing appeared to influence dialysis attendance, frequency of shortened treatments, and phosphorous and albumin levels favorably while less favorably impacting interdialytic weight gain. Changes in the HCCQ scores were not statistically significant from pre- to post-intervention, though the trend was in the direction of improvement in autonomy support, a main component of motivational interviewing. Patients and staff found the approach acceptable., Conclusions: Motivational interviewing shows promise as an intervention to improve adherence in adults undergoing hemodialysis.

Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. How arsenic is released from the sediment into the water remains poorly understood. Here, we show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite. We hypothesize that arsenic mobilization is a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice during weathering. Analysis of apatite from the source area in the Himalayan basin indicates the presence of elevated levels of arsenic, with an average concentration of 210 mg/kg. The rate of arsenic release is independent of the initial dissolved arsenic concentration and occurs at phosphate levels observed in Bangladesh aquifers. We also demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh aquifer sediments and groundwater. These results suggest that microbial mineral weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.

Background: A patient-based outcome measure with good measurement properties is urgently needed for use in clinical trials of foot surgery., Methods: We evaluated an existing foot pain and disability questionnaire (the Manchester Foot Pain and Disability Questionnaire) for its suitability as an outcome measure in the context of hallux valgus corrective surgery. Interviews with patients led to initial changes, resulting in 20 candidate questionnaire items with five response categories each. These were tested in a prospective study of 100 patients (representing 138 foot operations) undergoing hallux valgus corrective surgery. Analysis of underlying factor structure, dimensionality, internal reliability, construct validity and responsiveness of the questionnaire items in relation to (i) SF-36 general health survey and (ii) American Orthopaedic Foot & Ankle Society (AOFAS) hallux clinical scale resulted in a final 16 item questionnaire (the 'Manchester-Oxford Foot Questionnaire' (MOXFQ)), consisting of three domains/scales: 'Walking/standing' (seven items), 'Pain' (five items) and 'Social interaction' (four items) each having good measurement properties. All three domains were unidimensional., Conclusions: The new 16-item MOXFQ has good measurement properties in the context of outcomes assessment of surgery for hallux valgus. Future studies should assess the MOXFQ in the context of surgery for other foot and ankle conditions.

29 patients underwent 30 Scandinavian Total Ankle Replacements (STAR) by a single surgeon, over a four year period (1997 to 2001). There were 12 primary osteo arthritic patients with 6 post traumatic. The mean age of these 18 patients was 73. Eleven patients had rheumatoid arthritis and these had a mean age of 58 years at the time of surgery. One patient had bilateral replacements giving a total of 30 prostheses inserted. On review dorsiflexion had increased from a mean of 7 to 11 degrees while plantar flexion from 15 to 24 degrees. The American Orthopaedic Foot and Ankle hindfoot score was 95 (maximum 100) and the Kofoed ankle score revealed 17 excellent, 5 good and 4 fair. No patients had radiological evidence of loosening. Our intermediate term result for the STAR (W. Link GmbH & Co., Hamburg, Germany) ankle replacement has yielded 90% excellent and good functional outcomes in a mixed osteo arthritic and rheumatoid population which mirrors the results of those published from Scandinavia.

A method is described for continuous, nondestructive analysis of xylem-borne mineral nutrients in intact transpiring plants. The method uses the xylem-feeding insect the meadow spittlebug (Philaenus spumarius L. [Homoptera: Cercopidae]). This insect will feed from a wide range of plant species and organs. Insect excreta can be collected at all times of the day and night, and its mineral ion content can be analyzed rapidly, and without purification, by ion chromatography. The excreta will have a mineral content virtually identical to that of xylem sap. Cages suitable for containing the insects and collecting excreta from any desired location on plants in both laboratory and greenhouse are described. Even in the greenhouse, evaporation had only a minor effect on the sample ion content. Example results are presented which illustrate dynamics, over several days, in the xylem concentrations of sodium (Na(+)), potassium (K(+)), NH(4)(+), magnesium (Mg(2+)), calcium (Ca(2+)), chloride (Cl(-)), NO(3)(-), PO(4)(3-), and SO(4)(2-). These data were collected from young plants growing in pots of compost in the laboratory and from fully mature pepper (Capsicum annuum L. cv Bellboy) plants growing in hydroponics (rockwool) in the greenhouse. This method should facilitate studies of macronutrient uptake and transport in a range of plants and environments.

The relationship between ultrasonographic findings at birth and risk factors for developmental dysplasia of the hip have not been prospectively evaluated. Since implementing a routine screening programme for all new-born babies in 1989 we have collected 48 000 sets of data, including family history, birth presentation, mode of delivery and birth weight. Of the 92 babies (three per 1000 live births) with persistent ultrasonographic abnormality at 6 weeks only 20% displayed evidence of clinical instability at the original examination. Female babies without the additional risks of breech birth or positive family history were quantitatively the most significant group, accounting for 75% of cases treated. The majority of babies requiring intervention would not have been identified utilizing present criteria for selective ultrasound screening.

Human granulocytic ehrlichiosis (HGE) is a febrile tick-borne illness caused by a recently discovered intracellular bacterium remarkable for its tropism for professionally phagocytic neutrophils. Monoclonal antibodies against the P-selectin binding domain of the leukocyte P-selectin glycoprotein ligand, PSGL-1, prevented HGE cell binding and infection, as did enzymatic digestion of PSGL-1. Furthermore, simultaneous neoexpression in nonsusceptible cells of complementary DNAs for both PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII, allowed binding and infection by HGE. Thus, the HGE bacterium specifically bound to fucosylated leukocyte PSGL-1. Selectin mimicry is likely central to the organism's unique ability to target and infect neutrophils.

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with alpha1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.

Dapsone has clinical utility as an anti-inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits beta2 integrin (CD11b/CD18)-mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone-mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone-mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of beta2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant-induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses.

We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

Recent genetic and environmental theories of the etiology of homosexuality are evaluated. Interactive theory is proposed as a better alternative for understanding the development of sexual identity and sexual preference.

Objective: Our purpose was to review the indications for transabdominal cervicoisthmic cerclage to determine whether it is a valid alternative to transvaginal cerclage., Study Design: A retrospective review of transabdominal cerclage patients at one institution from 1978 to 1994, analysis of the indications for the transabdominal rather than the vaginal approach, and evaluation of fetal outcomes was performed., Results: Twenty-three patients underwent 24 transabdominal cerclages. The primary indication for transabdominal cervicoisthmic cerclage was failed transvaginal cerclage in 14 patients and anatomic unsuitability for transvaginal cerclage in nine. Of the latter, five were a result of diethylstilbestrol exposure and four a result of cervical surgery. All patients were successfully delivered of one or more live babies (total 28, including two sets of twins). Two losses occurred, one after rupture of membranes at 21 weeks on the second pregnancy after cerclage placement and one intraoperative loss with herniation of the membranes. The live birth rate was 93%, compared with 18% salvage of pregnancies beyond the first trimester before the transabdominal cervicoisthmic cerclage procedure. Complications included blood loss requiring transfusion (four patients), although none of these occurred in the last 12 patients., Conclusion: We conclude that all the patients had a history compatible with incompetent cervix requiring a cerclage, and none were suitable candidates for a vaginal cerclage. We further conclude that with strict indications transabdominal cervicoisthmic cerclage offers a high rate of fetal salvage with a minimum of complications in patients with extremely poor obstetric histories because of cervical incompetence.

The treatment and results of 13 blunt femoral fractures with an arterial injury were reviewed. Two of the 13 patients (15%) sustained segmental (two levels) arterial injuries. Stabilization of the femur fractures were performed before arterial repair in 10 of the 13 femurs. The results were determined at an average of 4.5 years. For the eight open fractures, two patients had above-knee amputations, no limb regained > 90 degrees of knee motion, four patients required a brace or cane, and three patients have intermittent wound drainage. The five closed fractures all regained full function with full knee motion. Due to the 15% incidence of segmental arterial injury, "wide-field" arteriography is advised for the evaluation of this injury. Femoral stabilization may be performed before arterial repair if the procedure does not encroach on the viability of the limb. The functional results depend on the soft-tissue damage, as illustrated by the poor results seen in open fractures.

Effects of low concentrations of detergents on cultured human foreskin keratinocytes were assessed in vitro. The viability and activity of keratinocytes were assessed by measuring reduction of a tetrazolium dye as an indicator of mitochondrial metabolism. The keratinocyte proliferative responses after incubation with detergents were assessed by a spectrophotometric assay employing crystal violet dye and a fluorometric assay determining total DNA content. Both the cationic detergent cetyltrimethylammonium bromide [CTAB] and the anionic detergent sodium lauryl sulfate [SLS] showed toxic effects on keratinocytes at concentrations as low as 3 micrograms/mg, but SLS was less toxic. However, both SLS and CTAB activated keratinocytes at very low concentrations. Proliferative activity and mitochondrial metabolism increased. Serum partially protected keratinocytes against toxic and stimulatory activities of both detergents. We suggest that detergents may directly damage keratinocytes and thereby produce irritant contact dermatitis, but activation of keratinocytes by low concentrations may also produce dermatitis, perhaps by causing keratinocytes to release cytokines.

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.

Distraction osteogenesis was used to treat five dogs with limb deformities or limb shortening. The affected bones underwent osteotomy, and modified external fixators were attached. Complications included pin loosening, implant breakage, and soft-tissue contracture. Adequate limb length was attained in all cases, but clinical results varied from poor to excellent. Two dogs were not lame after the procedure, two dogs had improved function but were still lame, and one dog had complications necessitating amputation.

Trichophyton rubrum infects skin. This fungus or its products might affect the function of epidermal cells. We previously reported that T. rubrum mannan (TRM) exhibits a suppressive effect on proliferation of human lymphocytes. The goal of the present study was to investigate the possibility of direct interaction of TRM with cultured normal human epidermal keratinocytes (EK). Mannan, a cell wall glycoprotein, was extracted from T. rubrum by precipitation with cetyltrimethylammonium bromide and conjugated with fluorescein isothiocyanate (FITC-TRM). After incubation of EK with 50 micrograms/ml FITC-TRM for 30 min, the surface of EK showed bright fluorescent staining. EK cultures pretreated with non-labelled TRM remained unstained. The fate of TRM bound to EK surface was determined in a time-course study. After pulse exposure to FITC-TRM, EK cultures were washed and incubated for various time periods. The EK moved surface mannan to the one area of cell membrane, so that at 4-6 h, the homogeneous staining of the entire cell surface was replaced by staining in a "cap" pattern. By 12 h, FITC-TRM was taken up into the cell, brought to the nuclear area and concentrated in the EK nucleoli. During the next 3 days nucleolar and cytoplasmic staining of the cells was observed. The intensity of fluorescence gradually diminished. On the 4th day, the sharp staining of organelles disappeared; instead, a large number of small fluorescent granules were seen intra- and extracellularly. By the 6th day after exposure, no EK staining remained. Thus, EK specifically bound, internalized and apparently catabolized TRM.(ABSTRACT TRUNCATED AT 250 WORDS)

We recently reported that the mannan component of Trichophyton rubrum cell wall (TRM) has an inhibitory influence on cell-mediated immune function in vitro. We now describe experiments designed to identify the target cell for this effect of TRM. T. rubrum mannan labeled with fluorescein (FITC-TRM) was incubated with peripheral blood mononuclear leukocytes, monocytes, or lymphocytes. Binding and uptake of the FITC-TRM were monitored by fluorescence microscopy and flow cytometry. Approximately 10% of mononuclear leukocytes were stained with this reagent and the fluorescent cells appeared to be monocytes by morphology. Virtually all purified monocytes and no purified lymphocytes stained with FITC-TRM. Flow cytometry to analyze FITC-TRM monocyte-specific binding of FITC-TRM involved the use of a phycoerythrin-labeled anti-CD14 antibody to identify monocytes. The only cells stained with FITC-TRM were those stained with the monocyte-specific antibody. The ability of monocytes to endocytose mannan was assessed by fluorescence microscopy. Cells were exposed to FITC-TRM and washed, and the staining pattern recorded periodically over a 48-h incubation period. After 15 min, staining was homogeneous and involved the entire cell surface; by 30 min, "patching" was observed; by 90 min, bright granules had formed along the cell border and a large number of small granules were present in the cytoplasm; by 8-12 h, the fluorescent granules were enlarged in size and reduced in number; by 24-36 h, the intensity of cytoplasmic fluorescence began to diminish; and, after 48 h, all fluorescent staining had disappeared. An additional feature of staining during the 8-12-h period was the appearance of a large round bright spot in the nuclear region of each cell, which may represent nucleolar staining. A role for "mannan receptors" is suggested by observations that FITC-TRM binding was prevented by unlabeled TRM or pretreatment of the monocytes with trypsin. Our finding that monocytes selectively and specifically bind TRM appears to identify the monocyte rather than the lymphocyte as the target cell for the inhibitory effect of mannan on cell-mediated immune function.

The anti-inflammatory influence of dapsone may involve suppression of neutrophil chemotaxis to selected attractants, but other actions of the drug are likely also involved. We have discovered that dapsone may suppress migration of neutrophils to extravascular sites through inhibition of adherence functions required for neutrophil recruitment. Neutrophil adherence mediated by integrins (CD11/CD18 or Mac-1 family receptors) was measured in vitro in terms of binding of stimulated cells to albumin-coated wells of microtiter plates, using phorbol myristate acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (FMLP) as stimuli. Adherence was assessed by staining attached cells with crystal violet dye and measuring the dye concentration at OD590 using an automated plate reader. The role of integrins in this assay was confirmed by the ability of anti-integrin antibody to suppress stimulated neutrophil adherence. The OD590 value for cells adhering to albumin in the absence of stimulus and dapsone averaged 0.2 +/- 0.04 (SEM) over five experiments. In the presence of 0.1 microM PMA or 10(-6) M FMLP, the OD590 values averaged 0.88 +/- 0.1 and 0.75 +/- 0.12, respectively. Dapsone did not affect unstimulated neutrophil adherence but, when present with stimulus, produced a dose-related inhibitory effect on adherence. Fifty percent inhibitory doses were approximately 150 micrograms/ml dapsone for both stimuli. Sulfapyridine reproduced the inhibitory effect of dapsone, but two structurally related compounds, hydrochlorothiazide and furosamide, did not. The observed ability of dapsone to inhibit neutrophil chemotaxis under agarose to FMLP and interleukin-8 may also be explained by interference with integrin-mediated adherence required for motility in this assay system. To consider if dapsone might have a similar inhibitory influence on neutrophil adherence in vivo, we tested the stimulated adherence function of neutrophils isolated from three individuals on dapsone therapy for dermatitis herpetiformis. Stimulated adherence of patients' cells averaged less than 40 percent of the control value. Suppression of leukocyte integrin function may therefore also contribute to the ability of dapsone to inhibit neutrophil infiltration in neutrophilic dermatoses.

Trichophyton rubrum causes 90% of chronic dermatophyte infections. Most patients with widespread chronic T. rubrum infection fail to express a delayed hypersensitivity reaction to intradermally injected trichophytin. We propose that cell-mediated immunity to T. rubrum may be suppressed in chronic infections by the mannan cell wall component of the fungus. The proposed suppressive effect of T. rubrum mannan on cell-mediated immunity was tested by measuring the ability of extracted mannan to inhibit lymphoproliferative responses of human mononuclear leukocytes to antigens, mitogens, and an anti-T-cell receptor antibody (anti-CD3) in vitro. Mannan was found to be highly antigenic in two of five donors and weakly antigenic in the other three. Despite its antigenic property, mannan exhibited a dose-related ability to inhibit lymphoproliferation stimulated by other agents including 1) antigens from Candida albicans, T. rubrum, and tetanus toxoid (ID50 = 250 micrograms/ml); 2) anti-CD3 antibody (ID50 = 250 micrograms/ml); and 3) Phaseolus limensis mitogenic lectin (ID50 = 64 micrograms/ml). Mannan added to cultures later than 24 h after initiation had no inhibitory influence, but culture of cells with mannan for a period of 24 h prior to the addition of stimulus enhanced the inhibitory effect of the glycoprotein. Lymphoproliferation in response to recombinant interleukin-2 (IL-2) was not inhibited. The influence of time of addition of mannan and the failure of mannan to inhibit IL-2-stimulated lymphoproliferation demonstrate that the suppressive effect of mannan must be pharmacologic rather than cytotoxic. The observed ability of T. rubrum cell wall mannan to suppress cell-mediated immune function in vitro may provide an important clue to a mechanism enabling the fungus to avoid elimination in chronically infected patients.

The antiinflammatory drug, phenylbutazone (PBZ), has been studied in terms of its influence on chemotactic deactivation of human neutrophils. When PBZ was present during the time of preincubation of cells with N-formyl-methionyl-phenylalanine (F-Met-Phe), loss of subsequent spontaneous mobility and chemotactic responsivity to F-Met-Phe did not occur. The action of PBZ to protect neutrophils from peptide-mediated chemotactic deactivation was found to involve in part its inhibitory influence on hexose monophosphate shunt activity and in part its antagonistic effect on interaction of peptide receptors with N-formyl peptide. Phenylbutazone interfered with binding of N-formyl-methionyl-leucyl-[3H]phenylalanine but not [125I]C5a to the neutrophil, displaced labeled tripeptide bound in the absence of PBZ, increased the dissociation constant (KD) for labeled tripeptide, and limited down regulation of peptide receptor function. These results provide an example of drug-mediated modulation of the interaction of neutrophils with N-formyl peptide and strongly support the possibility that PBZ interacts directly and specifically with the human neutrophil peptide receptor as a competitive antagonist. They also provide an additional example of a compound outside of th N-formyl peptide series that interacts with the peptide receptor.

A biologic tissue adhesive, a two-component fibrin sealant, for the immobilization of experimentally produced osteochondral fractures of the radial head and femoral condyle was investigated in dogs. The two components of the fibrin sealant were a sealer protein solution (fibrinogen) and a thrombin solution. A fibrinolytic inhibitor (C-esterase inhibitor) was added to prevent degradation of the fibrin by proteolytic enzymes. The operation was performed in 16 joints in four dogs. Control fractures on the right side were fixed with 5-cm Kirschner wires. No external immobilization was utilized. The dogs were killed at intervals of two, four, and eight weeks. There were no complications. All fractures healed uneventfully by eight weeks. Osseous repair seemed to be stimulated by the sealant and to occur faster than in the fractures fixed with pins.

A quantitative study of optic axons in the optic tracts of several species was performed to determine the number and percentage of optic axons that decussate. Animals were prepared by unilateral ocular enucleation, and light microscopic techniques were employed to count the fibers. The mean number of fibers projecting from a single eye was 732,119 for the pony, 1,041,739 for the cow, 534,755 for the sheep, and 442,629 for the pig. The mean degree of crossover at the chiasm was 80.8% in ponies, 82.9% in cattle, 88.9% in sheep, and 87.8% in pigs.

Light-producing reactions have been reported to occur after phagocytosis of opsonized particles by human polymorphonuclear neutrophils, eosinophils, and monocytes. Such chemiluminescence appears to be related to the generation of singlet oxygen, superoxide, and hydroxyl radicals, which have also been implicated as microbicidal agents. In examining the influences of various medium components on leukocyte chemiluminescence, we have observed that the amount of light measured is increased by addition of soluble protein, the amino acids tyrosine and tryptophane, or excess zymosan to the reaction medium. These agents appear to produce their effect not by increasing the rate of phagocytosis, but by providing substrate for secondary light-producing reactions. Polystyrene particles do not provide a suitable substrate for such secondary light-producing reactions. This is evidenced by the failure of ingested latex to stimulate high levels of chemiluminescence in the cellular system and their failure to augment light production in two noncellular chemiluminescent reactions. Some of the light generated in the cellular chemiluminescence response may derive from secondary reactions, which occur outside of the phagocyte. Support for this phenomenon is provided by two experiments. In one, addition of supplementary, nonopsonized zymosan to the reaction, after phagocytosis of opsonized zymosan is complete, resulted in an increased level of chemiluminescence. In another, addition of nonopsonized zymosan, together with latex particles, resulted in a significant increase in chemiluminescence. The results of the latter experiment also support our hypothesis that latex does not provide an appropriate substrate for secondary light-producing reactions. These observations suggest that leukocytes activated by phagocytosis generate electronically activated radicals which act intra- and extracellularly and that the amino acids tyrosine and trytophane may provide one substrate through which these agents act.

The efficacy of regular cervical examinations in detecting preterm labor, as well as possible risks, was studied for 133 preterm labor patients who had received regular cervical examinations as part of a preterm birth prevention program. When compared to all other patients experiencing preterm labor during the study period, the rates of preterm premature rupture of the membranes, chorioamnionitis, and postpartum endomyometritis were not increased. For 95 patients with preterm labor within 7 days of a routine cervical examination, labor onset was not temporally associated with the preceding examination. Preterm labor was diagnosed in 18 asymptomatic patients (18.2%) when the weekly examination revealed cervical change. Routine cervical exams, therefore, did not increase morbidity, and for some patients identified preterm labor before symptoms appeared.

We have investigated the mechanism of the nonspecific component of chemotactic deactivation of human neutrophils. This component of deactivation occurs on exposure of neutrophils to larger doses of cytotaxin and is expressed as depressed subsequent spontaneous migration and chemotaxis toward all cytotaxins tested. We demonstrate a possible mechanistic association between stimulation of hexose-monophosphate shunt activity and the nonspecific component of deactivation. This association is evidenced by failure to effect deactivation of 0 degrees C, by the ability of non-chemotactic stimulatory agents to mimic cytotaxin-induced deactivation, by the ability of agents which block shunt activity to protect against deactivation, and by the ability of methylene blue to deactivate neutrophils from individuals with chronic granulomatous disease. Thus, excessive stimulation of shunt activity, possibly through generation of products of oxygen metabolism, appears to have an inhibitory influence on nonspecific neutrophil migratory functions.

A system was described for studying bovine leukotaxis. Chemotaxis was readily observed toward bovine serum activated by zymosan of agarose. However, bacterial culture filtrates and peptides, which were potent chemotaxins for leukocytes from other species, failed to affect bovine leukotaxis. Using conditions suitable for studying binding to leukocytes from other species, specific binding of a radiolabeled chemotactic peptide to bovine leukocytes was not apparent. These data indicate that bovine leukocytes have chemotactic requirements distinct from those of other species.

A method was developed for collecting feline semen by electroejaculation combined with the use of a urethral catheter. The catheter facilitated handling the small volumes of semen for laboratory analysis. In 14 cats, semen volumes ranged from 0.019 to 0.284 ml (mean 0.076) and spermatozoa counts of ejaculates collected in the catheter ranged from 0.32 to 49.60 x 10(6) (mean 11.64 x 10(6)). Nine individuals were evaluated for retrograde ejaculation by quantitation of spermatozoa in pre-ejaculation and post-ejaculation urine samples. No spermatozoa were detected in pre-ejaculation samples but post-ejaculation urine samples contained from 0 to 11.88 x 10(6) (mean 4.55 x 10(6)) spermatozoa. The antegrade portion of the ejaculate contained from 6.3 to 100% (mean 59.1%) of the total number of spermatozoa ejaculated.

Candida albicans cell wall mannan polysaccharide has an ability to negatively influence cell-mediated immune function. We have attempted to identify the mechanism of this phenomenon by testing the modulatory effects of isolated mannan and the chemical catabolites of mannan on cell-mediated immune function in vitro. We have determined that mannan isolated by complexation with cetyltrimethylammonium bromide (CTAB) is more antigenic than mannan isolated by precipitation with copper and that CTAB mannan does not inhibit lymphoproliferation stimulated by another antigen. We have also determined that oligosaccharides of three sizes, derived by chemical catabolism of CTAB mannan, are not antigenic, but instead are immunoinhibitory. Immunoinhibition does not involve interference with the mitogenic activity of interleukin 2. A similar occurrence of oligosaccharides may be produced by catabolism of mannan in vivo as evidenced by the presence of oligosaccharides of similar size in cell-free supernatant fluids derived from mononuclear leukocytes incubated with tritiated mannan. We propose that catabolites of fungal mannan may contribute significantly to suppression of cell-mediated immunity in candidiasis.

A comparison was made of the sex roles of homosexual and heterosexual men and women on the Bem Sex Role Inventory, Personality Attributes Questionnaire, Personality Research Form Androgyny Scale, Adjective Checklist Masculinity and Femininity Scales, Extended Personality Attributes Questionnaire and Undesirable Characteristics Scale. The results indicated that homosexuals and heterosexuals differ in their response to different aspects of sex roles. The most consistent difference was the greater femininity of male homosexuals in respect to male heterosexuals. Other differences were scale-specific and the low interscale comparability indicated such scales should not be used interchangeably. Differences between results of studies comparing sex roles of the homosexuals and heterosexuals appear attributable to sample heterogeneity and distinctions between sex-role scales.