Estimating fat-free mass in elite-level male rowers: a four-compartment model validation of laboratory and field methods.

The purpose of this study was to investigate the accuracy of fat-free mass (FFM) estimates from two-compartment (2C) models including air displacement plethysmography (ADP), ultrasound (US), near-infrared interactance (NIR), and the Jackson and Pollock skinfold equation (SKF) against a criterion four-compartment (4C) model in elite male rowers. METHODS: Twenty-three elite-level male rowers (mean± SD; age 24.6 ± 2.2 years; stature: 191.4 ± 7.2 cm; mass: 87.2 ± 11.2 kg) participated in this investigation. All body composition assessments were performed on the same day in random order, except for hydrostatic weighing (HW), which was measured last. FFM was evaluated using a 4C model, which included total body water from bioimpedance spectroscopy, body volume from HW, and total body bone mineral via dual-energy X-ray absorptiometry. The major findings of the study were that the 2C models evaluated overestimated FFM and should be considered with caution for the assessment of FFM in elite male rowers. Future studies should use multiple-compartment models, with measurement of TBW and bone mineral content, for the estimation of FFM.

Adolescents' Understanding of Opioid-Induced Euphoria Measures and Proposed Measurement Revisions.

Prescription opioid misuse is an unintended consequence of acute pain management. Opioid-induced euphoria (OIE) with first therapeutic opioid exposure may influence opioid misuse. OIE is not assessed in clinical care and self-report measures of OIE have not been validated in adolescents. We (1) determined adolescents' ability to understand existing self-reported OIE measures, (2) revised measures for better understanding by this population, and (3) established initial content validity of revised measures with adolescents. Using runner's euphoria to simulate OIE in Study 1, 29 adolescents' (14 males) understanding of the Drug Effects Questionnaire (DEQ-5), the Addiction Resource Center Inventory Morphine Benzedrine Group scale (ARCI-MBG), and the ARCI Lysergic Acid Diethylamide scale (ARCI-LSD) were tested. In Study 2, 29 additional adolescents (9 males) participated in a modified Delphi study with focus groups to revise survey items to improve understanding by peers. In Study 1, runners understood <40% of ARCI-MBG and ARCI-LSD statements. In Study 2, all but 7 survey items were revised. Revised measures of OIE for adolescents may help define at-risk OIE phenotypes and validate risk assessments using survey methodology. Additional studies are needed to validate the revised OIE self-report measures with opioid-naive adolescents receiving opioids to treat acute pain.

Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Epidemiology of recurrent seizure disorders and epilepsy in cats under primary veterinary care in the United Kingdom.

BACKGROUND: Little epidemiological evaluation of recurrent seizure disorders in cats currently exists in veterinary literature. OBJECTIVES: To report the prevalence and risk factors for recurrent seizure disorders (RSD) and epilepsy in cats presented to primary care veterinary practices in the United Kingdom (UK). ANIMALS: A total of 285 547 cats under veterinary care during 2013 presenting to 282 primary care clinics in the UK. METHODS: Cohort study using multivariable logistic regression modeling for risk factor analysis. RESULTS: There were 458 confirmed RSD cases, giving a 1-year period prevalence of 0.16% (95% confidence interval [CI], 0.15-0.18). A subset of 114 (24.89%) cases was recorded as having epilepsy, giving a 1-year period prevalence of 0.04% (95% CI, 0.03-0.5). Increasing age was significantly associated with increasing odds of RSD. Breed, sex, neuter status, and body weight were not associated with RSD. Epilepsy was most frequently diagnosed in adult to middle-aged cats. Cats aged 3.0 to <6.0 years had 3.32 times higher odds of epilepsy diagnosis compared to cats <3.0 years of age. Insured cats were more likely to be diagnosed with epilepsy compared to noninsured cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Although less common than in dogs, RSD and epilepsy still comprise an important disorder group in the UK cat population. Aging is a significant risk factor for these disorders in cats.

Complement fixation by pemphigus antibody. V. Assembly of the membrane attack complex on cultured human keratinocytes.

Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.

The complement system in bullous pemphigoid. I. Complement and component levels in sera and blister fluids.

Compared with other serum and blister fluid proteins, total hemolytic complement was reduced in the blister fluid of six serologically positive bullous pemphigold patients while four serologically negative cases had blister fluid complement levels closely approaching the serum levels. Except for pemphigus vulgaris blisters. blister fluids from most patients with other bullous diseases and experimentally induced blisters had blister fluid complement levels more closely approaching the serum levels. With the exception of the two terminal components. C8 and C9, individual components of the complement sequence were also reduced in the blister fluids of the six bullous pemphigold patients with circulating basement membrane zone antibodies. On the other hand, transferrin and IgG levels of these same six serologically positive blister fluids closely approached the corresponding serum levels. Conversion of C3 proactivator was also demonstrable in the serologically positive bullous pemphigoid blister fluids, but not in the corresponding sera. Our studies, therefore, are suggestive of local activation of the complement sequence, by both the classical and alternate pathways, in blisters of serologically positive bullous pemphigold patients.

The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies.

IgE antibodies are usually thought to induce only immediate skin reactions. We have shown that the intradermal injection of a number of different allergens can produce a prolonged inflammatory reaction after the immediate wheal and flare in most sensitive subjects. This late inflammatory response occurs 6-12 h after challenge and is characterized by diffuse edema, erythema, pruritus, and heat. Both immediate and late responses can also be seen after passive sensitization of skin sites in nonatopic subjects. That IgE is involved in inducing the reaction was shown by the abolition of both immediate and late responses by passive transfer tests in the following experiments: (a) heating atopic serum at 56degreesC for 4 h, (b) removing IgE from the atopic serum by a solid phase anti-IgE immunoabsorbent, and (c) competitively inhibiting the binding of IgE antibodies to cells by an IgE myeloma protein. In addition, both responses were induced by affinity chromatography-purified IgE antibody, followed by antigenic challenge. Very similar lesions could also be induced by intradermal injection of Compound 48/80, thus suggesting a central role in the reaction for the mast cell or basophil. Histologically, the late phase is characterized by edema and a mixed cellular infiltration, predominantly lymphocytic but also containing eosinophils, neutrophils and basophils. Direct immunofluorescent staining did not show deposition of immunoglobulins or complement components, except IgM in 2 of 15 and C3 in 1 of 15 patients. This finding indicates that the late phase does not depend on the deposition of immune complexes. The results of the study suggest that IgE-allergen interaction on the surfaces of mast cells or on infiltrating basophils causes both immediate and late cutaneous responses.

The immunopathology of herpes gestationis. Immunofluorescence studies and characterization of "HG factor".

Nine skin biopsies from seven herpes gestationis patients were studied by immunofluorescence (IF) techniques. Basement membrane zone (BMZ) deposition of C3 and properdin was present in all nine skin specimens, while IgG deposition was apparent in only one. With in vitro C3 IF staining, positive BMZ staining (HG factor activity) was noted with all seven of our patients' serum samples tested. By standard indirect IF staining, however, only one of these serum samples contained BMZ antibodies of the IgG type. Two cord serum samples, tested by these same methods, yielded positive in vitro C3 staining (HG factor activity) but negative indirect IF staining (IgG). HG factor activity was found to be stable at 56 degrees C for 30 min and in two of three specimens at 56 degrees C for 1 h. Treatment of the complement source (normal human serum) used in the in vitro C3 staining assay with Mg2-EGTA or use of C2-deficient serum as the complement source inhibited HG factor activity. HG factor blocked the specific staining of the BMZ of normal human skin by labeled bullous pemphigoid antibodies. By sucrose density gradient ultracentrifugation and gel chromatography (Sephadex G-200), HG factor activity eluted with IgG-containing fractions. The highly purified IgG fraction of two herpes gestationis sera was also positive for HG factor activity. Our studies suggest that HG factor is an IgG antibody that may not be demonstrable by conventional IF methods, but which activates the classical complement pathway.

Complement activation in bullous skin diseases.

Pemphigus, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, and herpes gestationis are members of the chronic vesiculobullous skin diseases of man. The complement system, including both the classical and alternative pathways, may be important in the pathogenesis of these diseases. In pemphigus, early complement components (C1, C4, and C2) appear to be activated in addition to later components (C3 and C5), suggestive of classical pathway activation. Participation of properdin in addition to early complement components suggests local activation of both complement pathways in bullous pemphigoid and cicatricial pemphigoid. Herpes gestationis and dermatitis herpetiformis may be bullous skin diseases entirely mediated by the alternate or properdin pathway. The specific immunopathologic findings in these diseases are discussed.

The complement system in bullous pemphigoid: VII. Fixation of the regulatory protein beta 1H globulin by pemphigoid antibody.

Using in vitro complement immunofluorescent staining methods, serum samples from 5 active cases of bullous pemphigoid, with pemphigoid antibody titers of 320 or greater, were tested for their ability to fix the regulatory protein beta 1H globulin in addition to C4 and C3. All 5 samples yielded positive C3, C4 and beta 1H staining reactions in a linear fashion along the basement membrane zone. Heat inactivation or treatment of the complement source (fresh normal human serum) with EDTA, Mg2-EGTA abolished all 3 staining reactions. Substitution of C2-deficient serum as the source of complement inhibited both C3 and beta 1H staining but had no effect on C4 staining. Use of serum devoid of beta 1H (R beta 1H) minimally enhanced C3 staining while no beta 1H staining was observed. The addition of beta 1H to R beta 1H restored positive beta 1H staining. Skin biopsies of perilesional skin from 6 patients with bullous pemphigoid demonstrated heavy in vivo deposition of beta 1H in addition to C3. These studies suggest that pemphigoid antibodies will fix the regulatory protein beta 1H in addition to other complement components, a phenomenon which requires activation of the classical complement pathway and generation of the C3b amplification convertase.

Immunohistochemical studies of the human cutaneous basement membrane-anchoring fibril complex.

Antiserum to the human cutaneous basement membrane-anchoring fibril complex specifically binds to the basement membrane zone of normal human skin as demonstrated by indirect immunofluorescence and, in addition, cross-reacts with normal human glomerulus. This antiserum does not prevent binding of pemphigoid antibody to its specific antigen; thus the antigen of bullous pemphigoid is unlikely to be a component of the cutaneous basement membrane (basal lamina).

Complement fixation by pemphigus antibody. III. Altered epidermal cell membrane integrity mediated by pemphigus antibody and complement.

The present study investigates the effects of pemphigus IgG and complement upon cell viability and/or membrane integrity using trypan blue exclusion, ethidium bromide (EB) staining, and fluorescein diacetate (FDA) conversion by living cells. Forty-eight-hour cultivated epidermal monolayers of neonatal BALB/c mice were incubated in media containing 1 mg/ml purified pemphigus IgG for 48 h in either the presence or absence of complement (absorbed AB sera). Adherent and detached cells were examined by both phase and fluorescence microscopy. Results from trypan blue exclusion showed that pemphigus IgG plus complement produced a modest decrease in exclusion of the dye compared to pemphigus IgG without complement. When FDA/EB comparisons were made, however, the differences were more substantial. When complement plus pemphigus IgG was added to cultures, the number of FDA-positive adherent cells decreased significantly and the number of EB-positive detached cells increased significantly. The effects of complement were inhibited by the use of heat-inactivated AB sera or by C1q depletion of AB sera. No significant effect on the cells was observed in the presence or absence of complement when pemphigus F(ab')2 fragments or when normal IgG was used. Plasminogen depletion of the complement source did not interfere with complement and pemphigus IgG effects as judged by the FDA/EB assay. These studies suggest that pemphigus antibody in the presence of complement alters cell membrane integrity and supports the contention that complement may play a significant role in the mechanism of acantholysis.

Immunopathologic mechanisms in pemphigus and bullous pemphigoid.

Pemphigus and bullous pemphigoid are autoimmune bullous diseases of the skin. Pemphigus, an intraepidermal blistering disease, is characterized by autoantibodies reactive with antigens located in the intercellular spaces or on the surfaces of epidermal cells. These antibodies, which have recently been shown to activate complement, appear to be the cause of the basic pathologic process of pemphigus, acantholysis. The complement system and the plasminogen-plasmin system may be important mediators in the detachment of epidermal cells. Bullous pemphigoid, a subepidermal blistering disease, is characterized by autoantibodies reactive with an antigen located in the lamina lucida region of the basement membrane zone. These autoantibodies, which will avidly fix complement, appear to mediate subepidermal separation by attraction of a variety of inflammatory cells. Anaphylatoxins, released by activation of C4 and C3, or specific IgE antibodies, may activate mast cells with release of ECF-A attracting eosinophils. With activation of C5, C5a is released which could attract polymorphonuclear leukocytes. Antigen-specific lymphocytes, which can also contribute histamine releasing substances, may also be involved. The exact mechanism by which the epidermis separates from the dermis in bullous pemphigoid, however, remains unresolved.

Clq binding activity in lupus erythematosus: correlation with the "lupus band test".

A 131I C1q binding assay was employed to estimate C1q binding activity (C1q BA), presumably due to the presence of circulating immune complexes, in lupus erythematosus sera. Thirteen of 16 sera from patients with systemic lupus erythematosus (SLE) had elevated C1q BA. Only 1 of 17 sera from patients with generalized discoid LE (GDLE) and none of 5 localized DLE patient's sera had elevated C1q BA. A correlation between a positive LE band test and the level of C1q BA was apparent. A positive LE band test tended to occur in patients with higher levels of C1q BA, whereas a negative LE band test tended to occur in patients with low levels of C1q BA.

Complement fixation by pemphigus antibody. I. In vitro fixation to organ and tissue culture skin.

Although complement is often detected in the intercellular substance of pemphigus skin lesions, the ability of pemphigus antibodies to fix complement in vitro is controversial. The purpose of this study was to test in vitro complement fixation abilities of pemphigus antibodies further using organ and tissue culture methods. Epidermal cell monolayers from mouse tail were incubated with the purified IgG fraction of pemphigus serum followed by purified Clq. Binding of Clq, as well as IgG was demonstrated by immunofluorescence methods. When purified Clq was replaced with normal human serum as a complement source, positive C3 and C4 staining were also evident. When purified IgG of normal human serum was used in place of pemphigus IgG, similar immunofluorescence staining was not observed. Further evidence for complement fixation in vitro by pemphigus antibodies was obtained using organ cultures. Organ culture of normal human skin and monkey esophageal mucosa cultured in purified pemphigus IgG showed intercellular substance binding of IgG. No binding was observed when normal IgG was substituted for pemphigus IgG. Additional organ culture sections were then treated with complement (fresh normal human serum) and tested by in vitro complement staining. Fixation of Clq, C4, and C3 was noted in intercellular substance areas of organ cultured skin and mucosa incubated with pemphigus IgG but not those incubated with normal IgG. Prior treatment of pemphigus IgG organ cultured skin sections with unlabeled anti-C3, blocked positive C3 staining. These results suggest that some pemphigus antibodies are capable of activating complement in vitro.

Erythema multiforme: direct immunofluorescence studies and detection of circulating immune complexes.

The immunologic parameters of 23 patients with erythema multiforme who were seen by us (17 patients) or who had biopsies sent for immunofluorescence testing (6 cases) are reviewed. Biopsy specimens were sectioned and tested with labeled antisera to human IgG, IgA, IgM, C3 and fibrin. Fourteen biopsies showed IgM deposits in the superficial blood vessels, 13 demonstrated C3, 15 showed fibrin deposition, and 1 biopsy showed IgA deposition. All biopsies were negative for IgG. Eight serum samples tested by indirect IF were negative for skin-reactive antibodies. In addition to IF testing, serum samples from 20 patients were tested for circulating immune complexes with a Clq binding radioassay and a monoclonal rheumatoid factor (mRF) inhibition assay. Immune complexes were not detected by the Clq binding assay, but 6 of 20 serum samples demonstrated low to moderate levels of immune complexes by the mRF inhibition assay. By sucrose density gradient ultracentrifugation in the mRF-reactive material in one serum sample sedimented in high molecular weight fractions and also demonstrated anticomplementary activity. These findings suggest that immune complex formation and subsequent deposition in the cutaneous microvasculature may play a role in the pathogenesis of erythema multiforme.

Deposition of the membrane attack complex of complement in pemphigus vulgaris and pemphigus foliaceus skin.

The present study was performed to determine whether complement activation in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) results in the assembly of the terminal complement sequence or membrane attack complex (MAC) in skin lesions. Biopsy specimens of skin lesions from five patients with PV and three patients with PF contained C5, C7, C9, and the MAC related neoantigen (C5b-9 neoantigen) in intercellular substance areas (ICS), as well as IgG and the early complement components Clq, C4, and C3. The presence of these late complement components and the C5b-9 neoantigens in ICS sites of the skin lesions is indicative of complement activation by the pemphigus antibody, with subsequent assembly of the MAC. The binding of IgG and early complement components to ICS was observed in both non-lesional (normal appearing) skin and in skin lesions. However, no MAC could be detected in the normal appearing skin of our pemphigus patients. It was also noted that the MAC could be generated in vitro on cryostat sectioned normal human skin by pemphigus antibody in the presence of complement. Results of these studies suggest that complement activation may be related to membrane damage of epidermal cells in both PV and PF.

Inflammation in acne vulgaris: leukocyte attraction and cytotoxicity by comedonal material.

The chemoattraction of comedonal material for leukocytes was evaluated. Material from open comedones attracted mononculear leukocytes but did not attract polymorphonuclear leukocytes. At higher concentrations, comedonal material was cytotoxic for leukocytes of both types. Of the comedonal components tested, free fatty acids produced the greatest cytotoxicity. The attraction and killing of leukocytes by comedonal components may be the mechanisms for the initiation or the enhancement (or both) of inflammation in acne vulgaris.

Concurrent linear scleroderma and systemic lupus erythematosus: a report of two cases.

Two patients with linear scleroderma (en coup de sabre) developed systemic lupus erythematosus (SLE). This association has been well documented in only one previous case. The presence of high titer antibodies to ribonucleoprotein (RNP) initially led to the diagnosis of the mixed connective tissue disease. Development of more serious clinical involvement and antibodies to Sm (case 1) or native deoxyribonucleic acid (nDNA) (case 2) helped establish a diagnosis of SLE. Use of these studies in the differential diagnosis of systemic rheumatic diseases is disucssed briefly. The presence of anti-RNAP antibodies in patients with localized scleroderma may herald a more serious rheumatic disease.

Separation of epidermis from dermis with sodium thiocyanate.

After human skin is treated with 2 N sodium thiocyanate, epidermis is easily separated from dermis. The level of cleavage occurs at the lamina lucida of the basement membrane zone. Bullous pemphigoid antigen remains attached to the epidermis.