Weighted ensemble: Recent mathematical developments.

Weighted ensemble (WE) is an enhanced sampling method based on periodically replicating and pruning trajectories generated in parallel. WE has grown increasingly popular for computational biochemistry problems due, in part, to improved hardware and accessible software implementations. Algorithmic and analytical improvements have played an important role, and progress has accelerated in recent years. Here, we discuss and elaborate on the WE method from a mathematical perspective, highlighting recent results that enhance the computational efficiency. The mathematical theory reveals a new strategy for optimizing trajectory management that approaches the best possible variance while generalizing to systems of arbitrary dimension.

Classification, clinical manifestations, and immunopathological mechanisms of the epithelial variant of paraneoplastic autoimmune multiorgan syndrome: a reappraisal of paraneoplastic pemphigus.

BACKGROUND: Recent studies suggest that paraneoplastic pemphigus (PNP) is a heterogeneous autoimmune syndrome involving several internal organs and that the pathophysiological mechanisms mediating cutaneous, mucosal, and internal lesions are not limited to autoantibodies targeting adhesion molecules. OBJECTIVE: To classify the diverse mucocutaneous and respiratory presentations of PNP and characterize the effectors of humoral and cellular autoimmunity mediating epithelial tissue damage. METHODS: We examined 3 patients manifesting the lichen planus pemphigoideslike subtype of PNP. A combination of standard immunohistochemical techniques, enzyme-linked immunosorbent assay with desmoglein (DSG) baculoproteins, and an immunoprecipitation assay were used to characterize effectors of humoral and cellular autoimmunity in patients with PNP and in neonatal wild-type and DSG3-knockout mice with PNP phenotype induced by passive transfer of patients' IgGs. RESULTS: In addition to the known "PNP antigenic complex," epithelial targets recognized by PNP antibodies included 240-, 150-, 130-, 95-, 80-, 70-, 66-, and 40/42-kd proteins but excluded DSG1 and DSG3. In addition to skin and the epithelium lining upper digestive and respiratory tract mucosa, deposits of autoantibodies were found in kidney, urinary bladder, and smooth as well as striated muscle. Autoreactive cellular cytotoxicity was mediated by CD8(+) cytotoxic T lymphocytes, CD56(+) natural killer cells, and CD68(+) monocytes/macrophages. Inducible nitric oxide synthase was visualized both in activated effectors of cellular cytotoxicity and their targets. Keratin 14-positive basal epithelial cells sloughed from the large airways and obstructed small airways. CONCLUSIONS: The paraneoplastic disease of epithelial adhesion known as PNP in fact represents only 1 manifestation of a heterogeneous autoimmune syndrome in which patients, in addition to small airway occlusion and deposition of autoantibodies in different organs, may display a spectrum of at least 5 different clinical and immunopathological mucocutaneous variants (ie, pemphiguslike, pemphigoidlike, erythema multiforme-like, graft-vs-host disease-like, and lichen planus-like). We suggest that the more encompassing term "paraneoplastic autoimmune multiorgan syndrome," or PAMS, be applied. The pathophysiological mechanisms of PAMS involve both humoral and cellular autoimmunity responses. Epithelial cell membrane antigens other than DSG1 or DSG3 are targeted by effectors of PAMS autoimmunity. Apoptosis of damaged basal cells mediates epithelial clefting, and respiratory failure results possibly from obstruction of small airways with sloughed epithelial cells.

Identification and characterization of muscarinic acetylcholine receptor subtypes expressed in human skin melanocytes.

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (ml through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9,000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the ml, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Receptor-mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium influx and intracellular concentration.

Early stages of wound healing rely on the ability of keratinocytes (KCs) to move over the denuded dermis to re-epithelialize the defect. The agarose gel keratinocyte outgrowth system (AGKOS) is an in vitro model of skin re-epithelialization designed to study the migratory function of KCs. Endogenously secreted acetylcholine controls crawling locomotion of KCs in AGKOS by binding to the cholinergic receptors of both the nicotinic and the muscarinic classes that are expressed by KCs. In this study, we used AGKOS to elucidate the nicotinic pathway of cholinergic control of keratinocyte migration. Activation of the nicotinic acetylcholine receptors decreased the migration distance of KC in a dose-dependent fashion without altering cell viability. Nicotine also increased in a dose-dependent manner transmembrane influx of (45)Ca(2+), and caused a transient rise in the concentration of [Ca(2+)](i). Perfect correlation between concentration responses found in the migration and (45)Ca(2+) influx assays suggested that nicotine-induced inhibition of crawling locomotion relies on modulation of Ca(2+) metabolism in KCs. The effects of nicotine could be mediated by the alpha3- and the alpha7-containing nicotinic receptors visualized on KCs by immunostaining. Long-term incubation with nicotine up-regulated alpha7 and down-regulated alpha3 expression. Thus, nicotine exerts inhibitory effects on keratinocyte migration, and Ca(2+) serves as a second messenger in the signaling pathway. These results help explain deleterious effects of nicotine on wound re-epithelialization, and suggest that smoking may delay wound healing via nicotinic receptor-mediated pathway.

Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChRs) are expressed by human skin fibroblasts (HSF). We have identified the molecular subtypes of these receptors by reverse transcription-polymerase chain reaction (RT-PCR), using m1-m5 subtype-specific primers. These experiments showed that only mRNAs for m2, m4, and m5 mAChR subtypes are present in HSF. The RT-PCR products were characterized by restriction analysis and Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as immunogen corresponding to the C-terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranes of HSF in cell culture and specimens of normal human skin had a unique staining pattern specific for anti-m2 antibody, as well as for antibodies against m4 and m5. In Western blots of fibroblast proteins, the antibodies visualized the m2 receptor at 65 kDa, m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was examined by measuring muscarinic effects on intracellular free Ca2+ concentration ([Ca2+]i). Muscarine increased transiently [Ca2+]i in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine. Thus, the results of this study showed that HSF express m2, m4, and m5 mAChR subtypes, and that fibroblast mAChRs are coupled to the regulation of [Ca2+]i.

Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis.

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

Selective arterial embolization in the treatment of arterial priapism.

Arterial or 'high-flow' priapism is a rare complication of penile or perineal trauma. The case is reported of a patient with a more than 2-week history of priapism who was successfully treated by selective arterial embolization, with maintenance of potency.

Angiosarcoma of the penis.

Sarcoma of the penis is rare. We report an angiosarcoma that was essentially an incidental finding, despite the presence of metastatic lesions, in a 46-year-old man who presented with hematemesis and melena. The diagnosis of the primary lesion was confirmed by histology, and the presence of secondary lesions by fine needle aspiration cytology. The patient was given one dose of chemotherapy, but died of a myocardial infarction before being able to receive further treatment. The literature pertaining to this very unusual lesion is discussed.

Retrograde balloon dilatation for pelviureteric junction obstruction: long-term follow-up.

The results of 10 years' experience of treating pelviureteric junction (PUJ) obstruction by balloon dilatation are reviewed, and recommendations about the suitability of the technique for individual patients are made based on the patient's history and a preoperative DTPA renogram. Of 76 patients, 32 (42%) had no further symptoms after balloon dilatation. Six (8% continued to have mild loin pain only. In 33 patients (43%), there was no improvement in symptoms, split renal function, or drainage. Of this group, 21 patients (28%) underwent repeat balloon dilatation. Nine (12% became asymptomatic, and a further four (5%) had only minimal residual symptoms. The overall success rate of the procedure in terms of symptomatic abolition or improvement thus was 67%. Patients with < 25% function in the affected kidney preoperatively or who had undergone a previous pyeloplasty were the most likely to require additional treatment. No deaths were recorded, and morbidity was minimal.

Works in progress #7. Age-dependent fibrin clot invasion by human meniscal fibrochondrocytes. A preliminary report.

This study was performed to determine whether human fibrochondrocytes possess the same biologic potential for initiating a reparative response in a meniscal defect as that found in lower vertebrates. Small, circular defects were created in meniscal fragments obtained from nine men aged 14 to 72 years. The defects were filled with purified fibrin and placed in a culture medium. By 4 weeks, there was excellent cellular penetration into all clots. This study suggests that human meniscal fibrochondrocytes are also capable of initiating a reparative response, and the rapidity of this response appears to be age dependent. The 14- and 16-year-old menisci mounted a more rapid healing response in vitro than did those obtained from skeletally mature individuals.

Future directions. Collagen-based prostheses for meniscal regeneration.

Prosthetic meniscal replacement offers the ability to stabilize the meniscectomized knee and provide prophylaxis against early degenerative arthritis. Since prosthetic meniscal replacement may be performed in the setting of normal articular cartilage, a prosthesis will be required to match the exact joint configuration, induce the same lubricity, produce the same coefficient of friction, and absorb and dampen the same joint forces (without incurring significant creep or abrasion) as does the normal meniscus. This feat is currently beyond the capabilities of artificial materials alone. Alternatively, collagen-based prostheses acting as resorbable regeneration templates offer the possibility of inducing regrowth of new menisci. This paper presents a summary of hypotheses, considerations, and laboratory evidence for the use of collagen-based, resorbable matrices as regeneration templates.

In vitro culture of meniscal tissue.

The importance of the fibrocartilaginous menisci to the proper biomechanical function of the knee joint has been increasingly appreciated over the past 30 years. Meniscectomy is not the innocuous procedure it was once considered. Consequently, emphasis is now being placed on ways of repairing injured menisci in situ. To attain this goal, it is important to investigate the biology of the cells that synthesize and maintain the tissue that is to be repaired. In vitro culture techniques have aided in the understanding of how the cells of the meniscus (fibrochondrocytes) function and what is required to stimulate them to carry out the biologic functions they were designed to perform. In vitro culture of meniscal tissues may become an experimental tool for elucidating the requirements for meniscal repair and restoration of normal joint function after meniscal injury.

Pathology of the meniscus.

The pathologic description of the menisci is facilitated by grouping the various disorders into etiologic groups. These include congenital anomalies, traumatic conditions, inflammatory disorders, metabolic disorders, degenerative conditions, and neoplasms. In clinical practice, traumatic conditions, as exemplified by meniscal tears of the vertical-longitudinal or vertical-transverse type, are the most important pathologic disorders. Healing of meniscal tears occurs only when the tear involves the peripheral vascularized attachment of either the lateral or medial meniscus. Intensive clinical and animal experimentation suggests that serum-derived growth factors are necessary for meniscal healing. Chondrocalcinosis is extremely frequent in the menisci of older individuals and may or may not be associated with degenerative changes of the menisci, including fibrillation, loss of protein polysaccharide ground substance, and chondrocytic proliferation. The role of degenerative changes of the menisci, including tears of the horizontal type, in the development of osteoarthritis (OA) of the knee is a subject of intense interest, as is the relationship between chondrocalcinosis and OA. Neoplastic involvement of the menisci is essentially limited to invasion by tumors starting in or, more commonly, adjacent to the knee joints.

The ultrastructure and biochemistry of meniscal cartilage.

The meniscus is characterized at the light microscopic and ultrastructural levels by thick collagen fibers that are predominantly circumferential in orientation. The extracellular matrix of the meniscus is composed mainly of collagen, with smaller quantities of proteoglycans, matrix glycoproteins, and elastin. The collagen is predominantly Type I, with smaller quantities of Types II, III, and V. The proteoglycans are mainly large, aggregating proteoglycans with chondroitin sulfate as their dominant glycosaminoglycan. A small proportion of small dermatan sulfate proteoglycans is probably present. The matrix glycoproteins include the link proteins, the 116-k protein, and a group of adhesive or potentially adhesive proteins that includes Type VI collagen (here classified as a glycoprotein with a collagenous domain), fibronectin, and thrombospondin. The fibrochondrocytes of the meniscus appear to have considerable potential to respond to growth and other modulating factors in the repair or regeneration of the tissue.

An organ culture model for assaying wound repair of the fibrocartilaginous knee joint meniscus.

We describe an in vitro organ culture system that can be used to test the effect of various substances and compounds on the wound healing process in the fibrocartilaginous substance of the knee joint meniscus. Using culture medium containing either 10% fetal bovine serum (FBS) or our recently developed serum-free, defined culture medium (DM), we have demonstrated the ability of meniscal fibrochondrocytes from intact rabbit menisci to extricate themselves from their surrounding matrix and migrate into an exogenous, purified fibrin clot in vitro. After 4 weeks of culture in FBS-containing medium, the cells which had invaded the clot had initiated the early aspects of a typical reparative response; the same response did not occur in DM alone. Morphologically, the cells on the surface of the clot resembled the original superficial fibrochondrocytes, whereas those cells within the substance of the clot more closely resembled the original deep fibrochondrocytes. After 10 weeks, the reparative response had progressed only to a certain point and then failed to progress further under these culture conditions. However, use of this culture system should now make it possible to rapidly identify and quantitate those factors which would most likely be useful in continuing the reparative response and in affecting meniscal wound repair. Elucidation of the mechanisms and requirements for meniscal healing will eventually allow the practicing orthopaedic surgeon to effect in situ meniscal repair and obviate the need for meniscectomy and its morbid sequelae.

Cell culture of rabbit meniscal fibrochondrocytes II. Sulfated proteoglycan synthesis.

Rabbit meniscal fibrochondrocytes were grown in vitro under culture conditions previously shown to foster growth of this cell type. Regardless of the culture regimen employed, the cells synthesized sulfated proteoglycans which could be differentiated by their solubility when dialyzed against water. The water soluble proteoglycans (WSPG) were monomeric in nature and could be separated into sub-types based on their hydrodynamic size when analyzed by gel-filtration chromatography. The water insoluble proteoglycans (WIPG) appeared to represent hyaluronic acid-dependent aggregates of the larger of the two WSPG. The proteoglycans contained approximately 87% chondroitin sulfate and 5% dermatan sulfate. Keratan sulfate could not be detected. Addition of ascorbate to the culture medium did not change the amount or the hydrodynamic size of the proteoglycan aggregates but did alter the quantity of the larger WSPG monomer synthesized depending upon the culture regimen used. Thus, these cells are capable of expressing their differentiated phenotype in short-term monolayer cell culture.

Serum-free culture of rabbit meniscal fibrochondrocytes: proliferative response.

We have formulated a serum-free medium capable of supporting DNA synthesis in rabbit meniscal fibrochondrocytes at a level equivalent to 10% fetal bovine serum (FBS). The medium consists of a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 medium supplemented with transferrin (1 microgram/ml), selenium (1 pg/ml), trace metal mix (1:100), dexamethasone (100 ng/ml), insulin-like growth factors I and II (50 ng/ml each), pituitary fibroblast growth factor (100 ng/ml), and lactalbumin hydrolysate (2 micrograms/ml). Endothelial cell growth supplement could be substituted for lactalbumin hydrolysate to obtain similar results. Ventrex PC-1, a commercially available, low-protein, serum-free medium, was found to support proliferation of fibrochondrocytes but not as well as 10% FBS or our medium formulation. Lipid supplements, which are known to support the serum-free growth of hyaline chondrocytes, were found to be either of no value or antagonistic for the culture of fibrochondrocytes. Likewise, vitamin E alone, progesterone, putrescine, and hydrocortisone were also without benefit in our culture system. The cells had a more chondrocytic morphology when grown in defined medium as opposed to 10% FBS. The results of this study should now make it possible to identify and quantitate those factors necessary to affect meniscal repair by utilizing further techniques in vitro.

Aging phenomena and osteoarthritis: cause or coincidence? Claude P. Brown memorial lecture.

Osteoarthritis is an ubiquitous disease, primarily occurring in older individuals. Any attempt to explain the disease in terms of biologic aging must first define the basic pathology of the disease, accurately classify the clinicopathologic variants of the disease, and develop a scientific approach to the recognition of the pathologic physiology of any known precursor states. This epidemiologic information must insure comparability between populations of patients or animals under study, since significant differences in susceptibility among groups do exist. Cumulative environmental influences on joints do have pathophysiologic consequences but require careful study to isolate cause and effect from coincidental occurrences. Musculoskeletal aging itself is a complex process including both post-synthetic changes in the extracellular matrix macromolecules and alteration of phenotypic expression by the connective tissue cells themselves. In contrast to older assertions that chondrocytes are terminally differentiated cells incapable of replication, evidence has accumulated that chondrocytes proliferate in vivo and in vitro under appropriate conditions. Studies in several laboratories have confirmed that articular chondrocytes from aged animals both proliferate and synthesize macromolecules in a fashion similar to comparable cells extracted from young animals. Further studies are necessary before osteoarthritis can be definitively separated into aging-dependent and aging-independent categories.