Skin and respiratory ill-health attributed to occupational face mask use.

BACKGROUND: Face mask use in the workplace has become widespread since the onset of the Covid-19 pandemic and has been anecdotally linked to adverse health consequences. AIMS: To examine reports of adverse health consequences of occupational face mask use received by The Health and Occupation Research (THOR) network before and after the pandemic onset. METHODS: THOR databases were searched to identify all cases of ill-health attributed to 'face mask' or similar suspected causative agent between 1 January 2010 and 30 June 2021. RESULTS: Thirty two cases were identified in total, 18 reported by occupational physicians and 14 by dermatologists. Seventy-five per cent of cases were reported after the pandemic onset and 91% cases were in the health and social care sector. 25 of the 35 (71%) diagnoses were dermatological, the most frequent diagnoses being contact dermatitis (14 cases) and folliculitis/acne (6 cases). Of the seven respiratory diagnoses, four were exacerbation of pre-existing asthma. CONCLUSIONS: There is evidence of an abrupt increase in reports of predominantly dermatological ill-health attributed to occupational face mask use since the start of the pandemic. Respiratory presentations have also occurred.

Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans.

Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.

Macrolide-Resistant Mycoplasma pneumoniae in the United States as Determined from a National Surveillance Program.

There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 µg/ml, whereas MICs for MRMp were 16 to 32 µg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence.

Canine recommended breed weight ranges are not a good predictor of an ideal body condition score.

Breed-specific ideal bodyweight range information is widely used by dog owners and breeders as a guideline to ensure animals are within a healthy weight range. Body Condition Scoring, a method used by veterinarians to assess an animal's overall shape with regard to weight is considered to be an excellent method to determine an animal's overall body condition; these values, however, do not always correspond to published weight ranges. Here, the weight, neuter status, age and a nine-point Body Condition Score of a population of 140 purebred dogs were recorded and subsequently analysed to determine whether bodyweight was an effective predictor for Body Condition Scores. This comparison indicated that published recommended, breed-specific body weight ranges are not a good predictor for an ideal BCS and as such, guidelines for owners and breeders need to be systematically reviewed.

Characterizing zoo-housed Bactrian camel (Camelus bactrianus) reproduction using gonadal steroid metabolite analysis in feces.

Millions of people globally depend on camelids, which demands an increased knowledge of their reproduction. We used zoo-housed Bactrian camels (Camelus bactrianus) to better understand camelid reproductive physiology. Our specific objectives were to: 1) validate the use of fecal hormone metabolite analysis to characterize camel reproductive physiology during sexual maturity and pregnancy; and 2) determine the influence of season on male and female reproduction. We collected fecal samples from 1 male and 3 females housed at Lincoln Park Zoo (Chicago, IL, USA) 1 to 2 times per week for 3.5 years. Extracted hormones were analyzed using enzyme immunoassays for progestogen (FPM), estrogen (FEM), and androgen (FAM) metabolite concentrations. One female sexually matured during our study as evidenced by increased FEM baseline. Results demonstrated seasonal effects on male androgen production with FAMs higher (P < 0.05) January to June (mean ± SEM: 664.6 ± 22.6 ng/g wet feces), compared to July to December (401.6 ± 17.5 ng/g wet feces). One female experienced a persistent corpus luteum, a reproductive abnormality, which was identified by prolonged elevated FPM. FPMs increased during pregnancy for two females (452.9 ± 24.9 and 294.4 ± 19.8 ng/g wet feces) with a gestation of 404 d and 442 d, respectively. The third female never conceived. The FEMs varied (P < 0.05) during the year with no clear seasonal patterns (monthly mean range: 213.1-371.0 ng/g wet feces). Fecal hormone metabolite analysis is a validated method for assessing male seasonality and female pregnancy in the Bactrian camel and can for their management and conservation in zoos and the wild.

The Exocytosis-regulatory protein synaptotagmin VII mediates cell invasion by Trypanosoma cruzi.

The intracellular protozoan parasite Trypanosoma cruzi causes Chagas' disease, which affects millions of people in Latin America. T. cruzi enters a large number of cell types by an unusual mechanism that involves Ca(2+)-triggered fusion of lysosomes with the plasma membrane. Here we show that synaptotagmin VII (Syt VII), a ubiquitously expressed synaptotagmin isoform that regulates exocytosis of lysosomes, is localized on the membranes of intracellular vacuoles containing T. cruzi. Antibodies against the C(2)A domain of Syt VII or recombinant peptides including this domain inhibit cell entry by T. cruzi, but not by Toxoplasma gondii or Salmonella typhimurium. The C(2)A domains of other ubiquitously expressed synaptotagmin isoforms have no effect on T. cruzi invasion, and mutation of critical residues on Syt VII C(2)A abolish its inhibitory activity. These findings indicate that T. cruzi exploits the Syt VII-dependent, Ca(2+)-regulated lysosomal exocytic pathway for invading host cells.

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts.

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.

Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights.

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.

Project HELP: a study protocol to pilot test a shared decision-making tool about treatment options for patients with hepatitis C and chronic kidney disease.

BACKGROUND: Recent advances in treatment have given patients with chronic kidney disease (CKD) access to safer and more effective medications to treat comorbid hepatitis C virus (HCV) infection. Given the variety and complexity of treatment options that depend on patients' clinical characteristics and personal preferences, education and decision support are needed to prepare patients better to discuss treatment options with their clinicians. METHODS: Drawing on International Patient Decision Aids Standards guidelines, literature reviews, and guidance from a diverse expert advisory group of nephrologists, hepatologists, and patients, we will develop and test a HCV and CKD decision support tool. Named Project HELP (Helping Empower Liver and kidney Patients), this tool will support patients with HCV and CKD during decisions about whether, when, and how to treat each illness. The tool will (1) explain information using plain language and graphics; (2) provide a step-by-step process for thinking about treating HCV and CKD; (3) tailor relevant information to each user by asking about the individual's stage of CKD, stage of fibrosis, prior treatment, and comorbidities; (4) assess user knowledge and values for treatment choices; and (5) help individuals use and consider information appropriate to their values and needs to discuss with a clinician. A pilot study including 70 individuals will evaluate the tool's efficacy, usability, and likelihood of using it in clinical practice. Eligibility criteria will include individuals who understand and read English, who are at least 18 years old, have a diagnosis of HCV (any genotype) and CKD (any stage), and are considering treatment options. DISCUSSION: This study can identify particular characteristics of individuals or groups that might experience challenges initiating treatment for HCV in the CKD population. This tool could provide a resource to facilitate patient-clinician discussions regarding HCV and CKD treatment options.

Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype.

BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.

Extended low-dose dexamethasone suppression test for diagnosis of atypical Cushing's syndrome in dogs.

The purpose of this study was to evaluate extension of the low-dose dexamethasone suppression (LDDS) test from 8 h to 12 h to detect possible hypercortisolemia associated with atypical hyperadrenocorticism (AHAC). Twelve client-owned dogs were enrolled in the study: 6 healthy dogs (group 1) and 6 dogs with suspected AHAC (group 2). Baseline EDTA plasma samples were collected for endogenous ACTH determination using an immunoradiometric assay. Serum samples were collected before and at 4, 8, 10, and 12 h post-administration of 0.01 mg/kg dexamethasone IV for cortisol concentration determination via chemiluminescent assay. Mean endogenous ACTH concentration did not differ between groups (group 1: 22.4 pg/mL, group 2: 20.0 pg/mL; P > 0.2). Mean baseline cortisol concentration also did not differ significantly between groups (group 1: 3.03 µg/dL, group 2: 4.95 µg/dL; P > 0.2) nor was there any difference in mean cortisol concentration between the groups at any other time point (P > 0.2). The cortisol concentration from 1 dog in group 2 suppressed to 0.7 µg/dL at 8 h but increased to 1.5 µg/dL at 10 h and 3.7 µg/dL at 12 h post-dexamethasone. Based on results of this study, use of an extended LDDS test could not differentiate between healthy dogs and dogs with AHAC. Diagnosis of AHAC should continue to be based on prior established criteria until new testing has been identified.

Cryopreservation of animal oocytes and embryos: Current progress and future prospects.

Cryopreservation describes techniques that permit freezing and subsequent warming of biological samples without loss of viability. The application of cryopreservation in assisted reproductive technology encompasses the freezing of gametes, embryos, and primordial germ cells. Whilst some protocols still rely on slow-freezing techniques, most now use vitrification, or ultra-rapid freezing, for both oocytes and embryos due to an associated decreased risk of damage caused by the lack of ice crystal formation, unlike in slow-freezing techniques. Vitrification has demonstrated its use in many applications, not only following IVF procedures in human embryology clinics but also following in vitro production of embryos in agriculturally important, or endangered animal species, before embryo transfer. Here, we review the various cryopreservation and vitrification technologies that are used in both humans and other animals and discuss the most recent innovations in vitrification with a particular emphasis on their applicability to animal embryology.

Time-lapse embryo imaging and morphokinetic profiling: Towards a general characterisation of embryogenesis.

In vitro fertilisation is an effective method of assisted reproductive technology in both humans and certain non-human animal species. In most species, specifically, in humans and livestock, high in vitro fertilisation success rates are achieved via the transfer of embryos with the highest implantation and subsequent developmental potential. In order to reduce the risk of multiple gestation, which could be a result of the transfer of several embryos per cycle, restrictive transfer policies and methods to improve single embryo selection have been implemented. A non-invasive alternative to standard microscopic observation of post-fertilisation embryo morphology and development is time-lapse technology; this enables continuous, uninterrupted observation of embryo development from fertilisation to transfer. Today, there are several time-lapse devices that are commercially available for clinical use, and methods in which time-lapse could be used to improve embryology are continually being assessed. Here we review the use of time-lapse technology in the characterisation of embryogenesis and its role in embryo selection. Furthermore, the prospect of using this technology to identify aneuploidy in human embryos, as well as the use of time-lapse to improve embryological procedures in agriculturally important species such as the pig and cow are discussed.

Purified dietary n-3 polyunsaturated fatty acids alter diacylglycerol mass and molecular species composition in concanavalin A-stimulated murine splenocytes.

A low-dose, short-term dietary supplementation with highly purified (n-3) fatty acid ethyl esters was studied in mice to determine the effect on splenic cell membrane diacylglycerol mass and composition. Mice were fed diets containing either 3% safflower oil (SAF) ethyl esters, 2% SAF plus 1% eicosapentaenoic acid ethyl ester (EPA), or 2% SAF plus 1% docosahexaenoic acid ethyl ester (DHA). Following a 10-day feeding period, pathogen-free mice were sacrificed and splenic cells isolated and stimulated with concanavalin A (Con A) at 10 micrograms/ml. After 0 min (basal), 5 min, and 180 min, 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alkenyl-2-acyl-sn-glycerol subclasses were isolated and quantitated by HPLC. Diacylglycerol (DAG) was found to be the major diradylglycerol (DG) component in murine splenocytes. DHA-fed mice had significantly (P < 0.05) higher levels of DAG at all stimulation time points relative to EPA and SAF animals. Significant effects (P < 0.05) of diet, time, and a diet x time interaction (P < 0.05) were noted for various DAG molecular species. In general, a significantly higher (n-3) polyunsaturated fatty acid (PUFA) content in the EPA and DHA groups, and a significantly higher (n-6) PUFA content in the SAF group was noted. 18:0-22:5(n-3), 18:1-22:5(n-3) and 16:1-20:5(n-3) species were present only in EPA and DHA-DAG, confirming the incorporation of (n-3) fatty acids into splenocyte DAG. The data indicate that the molecular species composition of murine splenocyte DAG is significantly modulated by low-dose, short-term EPA and DHA feeding. In addition, substitution of SAF with DHA results in an increase in DAG mass. These alterations could potentially influence signal transduction pathways regulating lymphocyte function.

The changes in genetic and environmental variance with inbreeding in Drosophila melanogaster.

We performed a large-scale experiment on the effects of inbreeding and population bottlenecks on the additive genetic and environmental variance for morphological traits in Drosophila melanogaster. Fifty-two inbred lines were created from the progeny of single pairs, and 90 parent-offspring families on average were measured in each of these lines for six wing size and shape traits, as well as 1945 families from the outbred population from which the lines were derived. The amount of additive genetic variance has been observed to increase after such population bottlenecks in other studies; in contrast here the mean change in additive genetic variance was in very good agreement with classical additive theory, decreasing proportionally to the inbreeding coefficient of the lines. The residual, probably environmental, variance increased on average after inbreeding. Both components of variance were highly variable among inbred lines, with increases and decreases recorded for both. The variance among lines in the residual variance provides some evidence for a genetic basis of developmental stability. Changes in the phenotypic variance of these traits are largely due to changes in the genetic variance.

Inbreeding changes the shape of the genetic covariance matrix in Drosophila melanogaster.

The pattern of genetic covariation among traits (the G matrix) plays a central role in determining the pattern of evolutionary change from both natural selection and random genetic drift. Here we measure the effect of genetic drift on the shape of the G matrix using a large data set on the inheritance of wing characteristics in Drosophila melanogaster. Fifty-two inbred lines with a total of 4680 parent-offspring families were generated by one generation of brother-sister mating and compared to an outbred control population of 1945 families. In keeping with the theoretical expectation for a correlated set of additively determined traits, the average G matrix of the inbred lines remained proportional to the outbred control G matrix with a proportionality constant approximately equal to (1 - F), where F is the inbreeding coefficient. Further, the pattern of covariance among the means of the inbred lines induced by inbreeding was also proportional to the within-line G matrix of the control population with a constant very close to the expectation of 2F. Although the average G of the inbred lines did not show change in overall structure relative to the outbred controls, separate analysis revealed a great deal of variation among inbred lines around this expectation, including changes in the sign of genetic correlations. Since any given line can be quite different from the outbred control, it is likely that in nature unreplicated drift will lead to changes in the G matrix. Thus, the shape of G is malleable under genetic drift, and the evolutionary response of any particular population is likely to depend on the specifics of its evolutionary history.

Aromatase overexpression and breast hyperplasia, an in vivo model--continued overexpression of aromatase is sufficient to maintain hyperplasia without circulating estrogens, and aromatase inhibitors abrogate these preneoplastic changes in mammary glands.

To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors.

Studies of developing human hair shaft cells in vitro.

Methods for studying aspects of hair formation in vitro have been devised on the basis of isolating developing hair shaft cells. These cells were obtained using a sterile microdissection technique. Plucked anagen follicles were dissected free of surrounding tissues (inner and outer root sheaths), and presumptive hair shaft cells (including germinal epithelia) were cultured directly on mammalian fibroblasts or in media preconditioned by fibroblasts. Specimens were cultured either as dispersions or in whole tissue pieces. Trypsinized whole tissue specimens in culture were sometimes observed to form increased bulk, while dispersed cells appeared to elongate and form larger colonies. In sections of these colonies examined by transmission electron microscopy, intracellular hard keratin intermediate filaments (IFs) together with IF-matrix hard keratin complexes were observed. Radiolabelled cysteine [35S] was added to cultures (3-20 days), showing a continuing but reduced synthesis of hard keratin IF proteins (low-sulfur) over the period of study. Matrix protein (high-sulfur) production was drastically reduced after 3 days. Monoclonal antibodies directed against hair keratin IF components were used in Western transfers and immunofluorescent studies to help assess the specificity of proteins synthesized in culture. Our observations indicate that, with some refinement, the presently described methods enable preparation of hair shaft precursor cells suitable for observing certain hair-forming processes in vitro.

Axonal regeneration, but not myelination, is partially dependent on local cholesterol reutilization in regenerating nerve.

A recycling pathway in peripheral nerve permits cholesterol from degenerating myelin to be salvaged by macrophages and resupplied to myelinating Schwann cells by locally produced lipoproteins. A similar reutilization of cholesterol by regenerating axons has been proposed but not demonstrated. Neurites in culture, however, do take up cholesterol and cholesterol-containing lipoproteins, where these molecules are found to promote neurite extension. To test the requirement for cholesterol reutilization in axon regeneration and myelination, we examined 2 models of blocked intracellular cholesterol transport: 1) bone marrow transplants from Niemann-Pick C mice into wild-type recipient mice, and 2) imipramine treatment. Following nerve crush in these models, we found that unusually large, debris-filled macrophages appeared and persisted for many weeks. A morphometric analysis of regenerating nerves revealed that myelination proceeded at a normal rate (normal g-ratios), but that axon growth was retarded (decreased fiber numbers and diameters) in these animals. Cholesterol synthesis was elevated in these nerves, indicating that Schwann cells compensated for the decreased exogenous supply of cholesterol by up-regulating de novo synthesis to support myelination. These data indicate that Schwann cells are not dependent on cholesterol reutilization to support myelination, but that optimal axonal regeneration is dependent on a local supply of cholesterol.