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Unlabelled: Aerobic high-intensity interval training (HIT) improves cardiovascular capacity but may reduce the finite work capacity above critical power (W') and lead to atrophy of myosin heavy chain (MyHC)-2 fibers. Since whole-body vibration may enhance indices of anaerobic performance, we examined whether side-alternating whole-body vibration as a replacement for the active rest intervals during a 4 x 4 min HIT prevents decreases in anaerobic performance and capacity without compromising gains in aerobic function. Thirty-three young recreationally active men were randomly assigned to conduct either conventional 4 x 4 min HIT, HIT with 3 min of WBV at 18 Hz (HIT+VIB18) or 30 Hz (HIT+VIB30) in lieu of conventional rest intervals, or WBV at 30 Hz (VIB30). Pre and post training, critical power (CP), W', cellular muscle characteristics, as well as cardiovascular and neuromuscular variables were determined. W' (-14.3%, P = 0.013), maximal voluntary torque (-8.6%, P = 0.001), rate of force development (-10.5%, P = 0.018), maximal jumping power (-6.3%, P = 0.007) and cross-sectional areas of MyHC-2A fibers (-6.4%, P = 0.044) were reduced only after conventional HIT. CP, V̇O2peak, peak cardiac output, and overall capillary-to-fiber ratio were increased after HIT, HIT+VIB18, and HIT+VIB30 without differences between groups. HIT-specific reductions in anaerobic performance and capacity were prevented by replacing active rest intervals with side-alternating whole-body vibration, notably without compromising aerobic adaptations. Therefore, competitive cyclists (and potentially other endurance-oriented athletes) may benefit from replacing the active rest intervals during aerobic HIT with side-alternating whole-body vibration., Trial Registration: ClinicalTrials.gov Identifier: NCT01875146.

Introduction: Femoroacetabular impingement is a pathomechanical condition of the hip, which is often treated through arthroscopic techniques. The ensuing immobilization period is associated with decreases in muscle mass and bone mass. To date, minimal knowledge is present about the development of tissue mass during the considerably short rehabilitation period before returning to competition in elite endurance athletes., Case Description: Before and after surgery, a professional female Ironman triathlete underwent dual-energy X-ray absorptiometry and peripheral quantitative computed tomography measurements., Discussion and Evaluation: Areal bone mineral density (aBMD) of the proximal femur and lower extremity lean mass decreased in the surgically treated lower extremity during the two-month period of immobilization after the hip arthroscopy. These losses were compensated for after only six weeks of rehabilitation. A similar progression of aBMD values was observed in the lumbar spine. The adaptational pattern in volumetric BMD (vBMD) and volumetric bone mineral content (vBMC) of the tibiae were more complex, but attained pre-immobilization values for most variables also after six weeks of rehabilitation. All other variables attained pre-immobilization values no later than nine months after the surgical intervention., Conclusions: The athlete showed a high plasticity of bone and lean tissue with an optimal short- and midterm outcome. Following a two months immobilization period after a hip arthroscopy, aBMD, vBMD and vBMC achieved pre-surgical levels after four months of rehabilitation in a female Ironman triathlete. A nine-month follow-up measurement confirmed the safety of the fast return to sport.

Introduction: Endurance performance decreases during ageing due to alterations in physiological characteristics, energy stores, and psychological factors. To investigate alterations in physiological characteristics and body composition of elderly master athletes in response to an extreme endurance event, we present the case of the first ninety-year-old official male marathon finisher., Case Description: Before and directly after the marathon, a treadmill incremental test, dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, mechanography, and dynamometry measurements were conducted. The athlete finished the marathon in 6 h 48 min 55 s, which corresponds to an average competition speed of 6.19 km h(-1)., Discussion and Evaluation: Before the marathon, [Formula: see text] was 31.5 ml min(-1) kg(-1) body mass and peak heart rate was 140 beats min(-1). Total fat mass increased in the final preparation phase (+3.4%), while leg fat mass and leg lean mass were slightly reduced after the marathon (-3.7 and -1.6%, respectively). Countermovement jump (CMJ) peak power and peak velocity decreased after the marathon (-16.5 and -14.7%, respectively). Total impulse during CMJ and energy cost of running were not altered by the marathon. In the left leg, maximal voluntary ground reaction force (F m1LH) and maximal isometric voluntary torque (MIVT) were impaired after the marathon (-12.2 and -14.5%, respectively)., Conclusions: Side differences in F m1LH and MIVT could be attributed to the distinct non-symmetrical running pattern of the athlete. Similarities in alterations in leg composition and CMJ performance existed between the nonagenarian athlete and young marathon runners. In contrast, alterations in total body composition and m1LH performance were markedly different in the nonagenarian athlete when compared to his younger counterparts.

Purpose: It is a widely accepted premise in the scientific community and by athletes alike, that adding resistance exercise to a regular regimen of endurance training increases endurance performance in endurance-trained men. However, critical power (CP), capillarization, and myofiber size remain unaffected by this addition. Therefore, we tested whether the superimposition of resistance exercise with whole-body vibration and vascular occlusion (vibroX) would improve these variables in endurance-trained males relative to resistance exercise alone., Methods: Twenty-one young, endurance-trained males were randomly assigned either to a vibroX (n = 11) or resistance (n = 10) training group. Both groups trained in a progressive mode twice a week for 8 weeks. Pre and post training, histochemical muscle characteristics, thigh muscle size, endurance and strength parameters were determined., Results: vibroX increased CP (P = 0.001), overall capillary-to-fiber ratio (P = 0.001) and thigh lean mass (P < 0.001), while these parameters were unaffected by resistance training. The gain in CP by vibroX was positively correlated with the gain in capillarization (R(2) = 0.605, P = 0.008), and the gain in thigh lean mass was paralleled by increases in MyHC-1 and MyHC-2 fiber cross-sectional areas and strength. Maximum voluntary torque and the finite work capacity above CP (W') increased significantly only following resistance training., Conclusions: We achieved a proof of concept by demonstrating that modification of resistance exercise by superimposing side-alternating whole-body vibration and sustained vascular occlusion induced further improvements in CP, capillarization and hypertrophy, all of which were not observed with resistance training alone.

We assessed the prevalence and clinical features of genital skin changes in men after allogeneic hematopoietic stem cell transplantation (HSCT) and evaluated the correlation between genital chronic graft-versus-host disease (cGVHD) and other manifestations of cGVHD as well as sexual issues. In a cross-sectional cohort study, 155 male recipients alive 1 year or more after HSCT were assessed during their annual follow-up evaluation. Correlation between genital skin changes and other cGVHD manifestations was evaluated, and post-transplantation sexual contentment and sexual functioning were assessed by 2 self-assessment questionnaires, including the 5-item version of the International Index of Erectile Function (IIEF-5) and the modified Brief Sexual Symptom Checklist (mBSSC). Median time between HSCT and genital examination was 5.9 years (range, 1 to 30.3 years). Thirty-one of 155 patients (20%) presented with genital skin changes. Twenty-one of those (13%) presented clinically inflammatory genital skin changes considered as genital cGVHD: 12 had inflammatory (noninfectious) balanoposthitis, 6 had lichen sclerosis-like lesions, 5 had phimosis, and 2 patients had more than 1 feature. Patients with inflammatory genital skin changes had a significantly higher coincidence of oral (P < .0001), ocular (P < .002), and/or cutaneous cGVHD (P < .026) when compared with patients without genital lesions. The rate of IIEF-5 questionnaire response was 59% (91 of 155). Among them, 67% reported erectile dysfunction. Erectile dysfunction was significantly more frequent in patients with genital cGVHD (P = .0075). Seventy-five of 155 patients (48%) answered the mBSSC questionnaire. Only 40% of them reported sexual contentment. Genital skin changes in male recipients after allogeneic HSCT are frequent and seem to be an under-reported relevant late effect. Inflammatory genital skin changes can be considered as a form of genital cGVHD often associated with manifestations of extragenital mucocutaneous cGVHD., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Purpose: Triathletes lose body mass during an Ironman triathlon. However, the associated body composition changes remain enigmatic. Thus, the purpose of this study was to investigate Ironman-induced changes in segmental body composition, using for the first time dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT)., Methods: Before and after an Ironman triathlon, segmental body composition and lower leg tissue mass, areas and densities were assessed using DXA and pQCT, respectively, in eight non-professional male triathletes. In addition, blood and urine samples were collected for the determination of hydration status., Results: Body mass decreased by 1.9 ± 0.8 kg. This loss was due to 0.4 ± 0.3 and 1.4 ± 0.8 kg decrease in fat and lean mass, respectively (P < 0.01). Calf muscle density was reduced by 1.93 ± 1.04 % (P < 0.01). Hemoglobin, hematocrit, and plasma [K(+)] remained unchanged, while plasma [Na(+)] (P < 0.05), urine specific gravity and plasma and urine osmolality increased (P < 0.01)., Conclusions: The loss in lean mass was explained by a decrease in muscle density, as an indicator of glycogen loss, and increases in several indicators for dehydration. The measurement of body composition with DXA and pQCT before and after an Ironman triathlon provided exact values for the loss in fat and lean mass. Consequently, these results yielded more detailed insights into tissue catabolism during ultra-endurance exercise.

Background: The purpose was to investigate the effects of one dose of NaHCO3 per day for five consecutive days on cycling time-to-exhaustion (Tlim) at 'Critical Power' (CP) and acid-base parameters in endurance athletes., Methods: Eight trained male cyclists and triathletes completed two exercise periods in a randomized, placebo-controlled, double-blind interventional crossover investigation. Before each period, CP was determined. Afterwards, participants completed five constant-load cycling trials at CP until volitional exhaustion on five consecutive days, either after a dose of NaHCO3 (0.3 g·kg-1 body mass) or placebo (0.045 g·kg-1 body mass NaCl)., Results: Average Tlim increased by 23.5% with NaHCO3 supplementation as compared to placebo (826.5 ± 180.1 vs. 669.0 ± 167.2 s; P = 0.001). However, there was no time effect for Tlim (P = 0.375). [HCO3-] showed a main effect for condition (NaHCO3: 32.5 ± 2.2 mmol·l-1; placebo: 26.2 ± 1.4 mmol·l-1; P < 0.001) but not for time (P = 0.835). NaHCO3 supplementation resulted in an expansion of plasma volume relative to placebo (P = 0.003)., Conclusions: The increase in Tlim was accompanied by an increase in [HCO3-], suggesting that acidosis might be a limiting factor for exercise at CP. Prolonged NaHCO3 supplementation did not lead to a further increase in [HCO3-] due to the concurrent elevation in plasma volume. This may explain why Tlim remained unaltered despite the prolonged NaHCO3 supplementation period. Ingestion of one single NaHCO3 dose per day before the competition during multiday competitions or tournaments might be a valuable strategy for performance enhancement., Trial Registration: Trial registration: ClinicalTrials.gov Identifier NCT01621074.

Vitamin D is important for bone health, with low vitamin D levels being associated with skeletal fragility and fractures. Among its other biological activities, 1,25-dihydroxyvitamin D (1,25(OH)(2) D), stimulates the in vitro differentiation of human marrow stromal cells (hMSCs) to osteoblasts, which can be monitored by increases in alkaline phosphatase enzyme activity or osteocalcin gene expression. In this study, we tested the hypotheses that age and clinical attributes of subjects influence in vitro responsiveness of hMSCs to 1,25(OH)(2) D(3) . In a cohort of subjects whose hMSCs were isolated from bone marrow discarded during hip replacement surgery for osteoarthritis, there were significant inverse correlations with age for bone mineral density, renal function, body mass index, fat mass index, and lean mass index (n = 36-53). There were significant correlations with serum 25(OH)D for serum parathyroid hormone (PTH), body mass index, fat mass index, and lean mass index (n = 47-50). In vivo-in vitro correlation analyses indicated that there were significantly greater in vitro effects of 1,25(OH)(2) D(3) to stimulate osteoblast differentiation in hMSCs obtained from subjects who were younger than 65 years of age, or who had serum 25(OH)D ≤ 20 ng/mL, elevated serum PTH, or better renal function, assessed by estimated glomerular filtration rate. The greater in vitro stimulation of osteoblast differentiation by 1,25(OH)(2) D(3) in hMSCs from vitamin D-deficient subjects suggests that vitamin D replenishment may lead to more vigorous bone formation in subjects at risk., (Copyright © 2012 American Society for Bone and Mineral Research.)

Background: HIV-1 protease is subjected to dual selection pressure exerted by protease inhibitors (PIs) and cytotoxic T lymphocytes (CTL). Recently, we identified KMIGGIGGF (KF9) as a HLA-B*1501-restricted CTL epitope, including several major PI resistance mutations (M46I/L, I47A/V, G48V, I50V). To assess potential interactions between KF9-specific CTL and emergence of these important resistance mutations, we studied CTL recognition of the mutations and analyzed protease sequences in an HLA-I–typed patient cohort., Methods: CTL recognition of KF9 and resistance mutations in KF9 were studied in 38 HLA-B*1501-positive HIV-1–infected patients using variant KF9 peptides in interferon-g enzyme-linked immunospot assays. Protease sequences were analyzed in 302 HLA-I–typed HIV-1–infected patients., Results: G48V abolished KF9 recognition by CTL in all patients. Furthermore, M46I, I47A, and I50V could impair or abolish CTL recognition in many patients. In contrast, M46L and I47V showed good CTL recognition in nearly all patients. HIV-1 protease sequence analysis showed no statistical correlation between the occurrence of resistance mutations in KF9 and HLA-B*1501. Viral load in patients failing therapy with KF9 mutations was significantly lower in HLA-B*1501-positive patients in comparison with HLA-B*1501-negative patients., Conclusions: PI mutations, G48V, M46I, and I47A, can abrogate CTL recognition, indicating potential interactions between development of drug resistance and CTL response. However, we could not find evidence that development of these PI mutations is influenced by KF9-specific CTL.

Cryopyrin-associated periodic fever syndrome (CAPS) is a rare inherited disease due to mutations in the NLRP3 (also called CIAS1) gene on chromosome 1q44 resulting in overproduction of interleukin-1. CAPS comprises three clinically overlapping disorders including Muckle-Wells syndrome. We report on two half-siblings with Muckle-Wells syndrome who were successfully treated with the interleukin-1 beta antibody canakinumab. Despite reduced dosing and longer treatment intervals compared to the recommended dosing schedule (e.g. 150 mg every 8 weeks), the efficacy and tolerability of canakinumab was impressive in both patients. The pharmacologic properties of canakinumab are reviewed and the clinical and economical aspects highlighted. We show that with individualized 'reflare-guided' administrations of canakinumab overall costs could hypothetically be reduced by 50% (approx. USD 60,000/patient/year) and therefore could have a major impact on treatment costs., (Copyright © 2011 S. Karger AG, Basel.)

Introduction: HIV type-1 (HIV-1) protease (PR) and cleavage site (CS) mutations accumulate in protease-inhibitor-resistant isolates. HIV-1 CS mutation 431V is the most frequent treatment-associated CS mutation; however, little is known about its origin in treatment-naive HIV-1 isolates. Recently, it has been shown that the CS mutation 431V is located within the human leukocyte antigen (HLA)-B*13-restricted cytotoxic T-lymphocyte (CTL) epitope RQANFLGKI (RI9). Therefore, we investigated whether the presence of CS mutation 431V might additionally be related to immune escape., Methods: CTL recognition of RI9 and of RI9 variants carrying the 431V or the 436R mutation was analysed by ELISPOT in nine HLA-B*13-positive HIV-1-infected patients. Treatment-naive HIV-1-infected patients with primary drug-resistant HIV-1 isolates (n=58) or carrying 431V (n=4) were genotyped for HLA class I alleles., Results: ELISPOT analysis showed different patterns of CTL recognition of RI9. CS mutation 431V could abrogate recognition by RI9-specific CTL in a subgroup of patients. Nevertheless, in our study, the occurrence of 431V in treatment-naive HIV-1 without primary drug resistance could not be explained by HLA-B*13-mediated immune selection. In patients with primary drug-resistant HIV-1 isolates, the frequency of HLA-B*13 was not increased and HLA-B*13 did not correlate with CS mutations 436R or 431V., Conclusions: HIV-1 CS mutation 431V can abrogate CTL recognition, indicating interactions between development of drug resistance and the CTL response. However, we could not find evidence that the presence of 431V in treatment-naive HIV-1 isolates with and without primary drug resistance is related to immune selection by HLA-B*13 or other HLA class I alleles.

Objectives: To study the role of cytotoxic T-lymphocyte (CTL) escape for disease progression in HIV-1 infection, we analyzed the CTL response to the dominant human leukocyte antigen (HLA)-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef., Methods: HIV-1 nef genes derived from 56 patients were analyzed by polymerase chain reaction (PCR)-based sequencing. T-cell responses against FL8 and mutated FL8 variants were detected by gamma-interferon (gamma-IFN) enzyme linked immunospot (ELISPOT) assay., Results: The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1-infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition; however, there was strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 cell counts and higher viral loads., Conclusions: Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 cell counts indicates an important role of CTL escape mutations for disease progression.

Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8(+) T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R(264)K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R(264)K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R(264)K mutation was associated in vivo with the development of a rare secondary mutation, S(173)A, which restored viral replication in vitro. Furthermore, infectivity of the R(264)K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R(264)K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R(264)K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.

vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55(gag). Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen, alpha-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101-112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with alpha-actinin 1. These findings suggest that the interaction with alpha-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

The highly conserved Vpr protein mediates cell cycle arrest, transcriptional transactivation, and nuclear import of the preintegration complex in human immunodeficiency virus type 1. To identify functional domains in simian immunodeficiency virus (SIV) mac239 Vpr, we mutagenized selected motifs within an alpha-helical region and two C-terminal HxRxG motifs. All Vpr mutants located to the nucleus. Substitution of four amino acids in the alpha-helical domain did not interfere with cell cycle arrest, while a single substitution abolished cell cycle arrest function. Mutation of the first HxRxG motif to AxAxA also resulted in loss of cell cycle arrest, while mutation of the second motif had no effect. Interestingly, both Vpr mutants impaired in cell cycle arrest function also showed reduced transactivation of the SIV long terminal repeat, suggesting that arrest of cells at G(2)/M mediates or contributes to transactivation by Vpr.

Studies with human and animal culture systems indicate that a sub-population of bone marrow stromal cells has the potential to differentiate into osteoblasts. There are conflicting reports on the effects of age on human marrow-derived osteogenic cells. In this study, we used a three dimensional (3D) culture system and quantitative RT-PCR methods to test the hypothesis that the osteogenic potential of human bone marrow stromal cells decreases with age. Marrow was obtained from 39 men aged 37 to 86 years, during the course of total hip arthroplasty. Low-density mononuclear cells were seeded onto 3D collagen sponges and cultured for 3 weeks. Histological sections of sponges were stained for alkaline phosphatase activity and were scored as positive or negative. In the group < or = 50 years, 7 of 11 samples (63%) were positive, whereas only 5 of 19 (26%) of the samples in the group > or = 60 years were positive (p = 0.0504). As revealed by RT-PCR, there was no expression of alkaline phosphatase or collagen type I mRNA before culture, however there were strong signals after 3 weeks, an indication of osteoblast differentiation in vitro. We performed a quantitative, competitive RT-PCR assay with 8 samples (age range 38-80) and showed that the group < or = 50 years had 3-fold more mRNA for alkaline phosphatase than the group > or = 60 years (p = 0.021). There was a significant decrease with age (r = - 0.78, p = 0.028). These molecular and histoenzymatic data indicate that the osteogenic potential of human bone marrow cells decreases with age., (Copyright 2001 Wiley-Liss, Inc.)

With advancing age, an increase in bone resorption relative to bone formation results in bone loss. Bone marrow stromal cells and their products support osteoclastogenesis from hematopoietic progenitors. Another of their products, osteoprotegerin (OPG), blocks the osteoclast-stimulatory effects of OPG ligand. We tested the hypothesis that with advancing age there is a decrease in OPG expression by human bone marrow cells. Bone marrow cells were obtained from 18 subjects (age range 38-84 years). Expression of mRNA transcripts of OPG was assessed by quantitative competitive RT-PCR. Median number of OPG transcripts in the younger group was 0. 3 zetptomoles (range 0.01 to 1.30) and was higher than in the older group's median of 0.06 (range 0 to 0.5; p < 0.05). The decline in the expression of OPG with age may increase the capacity of stromal/osteoblast cells to support osteoclastogenesis., (Copyright 2000 Academic Press.)

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.

Many types of injuries to the meniscus of the knee joint result in defects that do not heal, leading to pain and dysfunction. Several ongoing investigations are developing porous absorbable matrices to be used alone or seeded with cultured cells to facilitate regeneration of this tissue. The objective of this study was to evaluate in vitro the contractile behavior of meniscal cells seeded in type I and type II collagen matrices. In many connective tissues, fibroblasts that have assumed a contractile phenotype (myofibroblasts) have been found to play an important role in healing and in pathological conditions. This phenotype, if expressed by meniscal cells, could affect their behavior in cell-seeded matrices developed for tissue engineering. In this study, the presence of a contractile actin isoform, alpha-smooth muscle (alpha-SM) actin, was assessed by immunohistochemistry in normal calf meniscal tissue and in meniscal cells in 2- and 3-dimensional culture. Calf meniscus cells were seeded in type I and type II collagen-glycosaminoglycan (GAG) matrices. The diameter of the matrices was measured every 2-3 days. Immunohistochemical staining of the 2-dimensional cultures for alpha-SM actin was performed after 1, 3, and 7 days and the staining of the seeded matrices was at 1, 7, 14, and 21 days. Transmission electron microscopy (TEM) was performed on selected samples. After 3 weeks the seeded type I matrices displayed a significant shrinkage of almost 50% whereas the type II matrix and both types of unseeded controls showed almost no contraction over the same time period. Positive staining for the alpha-SM actin phenotype was seen in 10% of the cells of the normal tissue but was present in all cells seeded in monolayer and in both types of matrices. TEM of representative cell-seeded matrices showed microfilaments approximately 7 nm thick, consistent with the myofibroblast phenotype. This is the first report of alpha-SM actin containing cells in the knee meniscus. The finding that, under certain conditions, meniscal cells can express the myofibroblast phenotype warrants study of their role in meniscal healing and the tissue response to implants to facilitate tissue regeneration., (Copyright 1999 John Wiley & Sons, Inc.)

The objective of this study was to determine the proliferative and biosynthetic activity of calf meniscus cells seeded in type I and type II collagen-glycosaminoglycan (GAG) copolymers with the overall goal to develop a cell-seeded implant for future investigations to improve the regeneration of the knee meniscus. The cell-seeded matrices were digested in protease and analyzed for GAG by a modification of the dimethyl-methylene blue method and assayed for DNA content. Other specimens were evaluated histologically after 1, 7, 14 and 21 days. Contraction of the same types of matrices, seeded with adult canine meniscus cells, was measured at the same time points. After three weeks, cells were observed throughout the type II matrix, whereas the type I matrix was densely populated at the margins. The cell morphology and the cell density after three weeks in both matrices was consistent with the normal meniscus. DNA assay for the type I matrix showed a 40% decrease over the first week and a final amount of DNA that was not significantly different from the initial value, whereas the type II matrix doubled its DNA content over the same time period. The cells continued their biosynthesis of GAG and type I collagen. GAG content of the type II matrix increased by 50% more than the type I matrix after three weeks. Over the same time period, the type I matrix displayed a significant shrinkage to approximately 50% of its initial value whereas in contrast, the type II matrix and the unseeded controls showed no significant shrinkage. The number of cells and the higher GAG synthesis in the type II matrix, and its resistance to cell-mediated contracture, commend it for future investigation of the regeneration of meniscus in vivo.

The objective of this study was to investigate the presence of a contractile actin isoform, alpha-smooth muscle actin, in annulus fibrosus cells in situ and in two and three-dimensional cultures. Annulus fibrosus cells were isolated from healthy adult dogs, serial passaged, and then injected into porous collagen-glycosaminoglycan copolymers consisting of either type-I or type-II collagen. Alpha-smooth muscle actin was detected in the cells in tissue samples and in culture by immunohistochemistry. The number of cells and glycosaminoglycan content of the matrices were determined after 1, 7, and 14 days, and the diameters of the specimens were measured every 2 days. Although few annulus fibrosus cells in vivo displayed the presence of the alpha-smooth muscle actin isoform, most cells in two-dimensional culture demonstrated this phenotype. The contractile behavior of these cells was shown by the cell-mediated contraction of type-I collagen-glycosaminoglycan scaffolds after 8 days in culture. Glycosaminoglycan production was not significantly different in the seeded type-I matrices than in the unseeded matrices, whereas the seeded type-II matrices had a significant increase in glycosaminoglycan production between days 1 and 14 compared with the unseeded controls. This is the first report of both the expression of the contractile alpha-smooth muscle actin isoform in intervertebral disc cells and the ability of the cells to contract a collagen matrix. This finding could aid in better understanding the nature of cells in the annulus.

The purpose of this study was to examine vascular structural alterations longitudinally in spontaneous and Dahl genetic hypertension. Hypertensive and control animals were studied at 5,9-11, and 17-19 weeks of age to permit analysis of prehypertensive, early and established hypertensive stages. Minimal and maximal resistance of the hindquarter vasculature was used as a functional assessment of structural alterations. At 5 weeks of age, the minimal vascular resistance of the spontaneously hypertensive rats (SHR) was elevated over Wistar-Kyoto (WKY) (p less than 0.02) but there was no difference between Dahl salt-sensitive (S) and resistant (R) rats. In both sets of animals, the minimal vascular resistance of the hypertensive group was significantly elevated over controls with age: p less than 0.001 in SHR; p less than 0.001 in Dahl S. The maximal vasoconstrictor response was significantly greater with age in SHR than in WKY, (p less than 0.001), but was not different in Dahl S compared to R. Thus, structural alterations, determined by assessing minimal vascular resistance, are present in both spontaneous and Dahl salt-sensitive hypertension, but the origin of the two differ. An increase in smooth muscle mass, assessed by maximal constriction, contributes importantly to the structural alterations in spontaneous hypertension; in Dahl S, other factors appear to contribute to structural alterations. Further, structural alterations precede frank hypertension in SHR but not in Dahl S hypertension.

Eleven patients had neurologic symptoms after taking "look-alike" pills thought to contain amphetamines but actually containing phenylpropanolamine. Phenylpropanolamine, a nonregulated drug, is structurally and functionally similar to amphetamines. Acute onset of headache, psychiatric symptoms, or seizures in young patients may be due to phenylpropanolamine use.

The hypothesis of this study is that the chronic presence of intact sympathetic innervation influences the structure, and thereby function, of the rabbit ear vascular bed, and that this influence may be age related. Therefore we examined the effect of chronic sympathetic denervation on vascular resistance of the rabbit ear nine to ten weeks after unilateral superior cervical ganglionectomy. Two age groups were studied: growing juvenile rabbits denervated at 4 weeks of age and adult rabbits denervated at 16 weeks. We assessed the flow-pressure (resistance) curves of the chronically denervated and contralateral innervated isolated rabbit ears during maximum dilation. The flow-pressure curve was significantly shifted downward in the denervated compared to the contralateral innervated ears of juvenile rabbits operated on at 4 weeks (p less than 0.001) but was not different in adult rabbits operated on at 16 weeks. These results support the concept that the sympathetic nerves chronically influence blood vessel properties in growing animals, apart from their acute effect on vasomotor tone.

We recently investigated the effect of chronic sympathetic denervation on the intact ear vasculature of normotensive rabbits and found an important long-term or trophic influence of sympathetic nerves on vascular structural properties. To determine the effect of coarctation hypertension on this relationship, we studied 12 wk old rabbits 8 wk after a unilateral superior cervical ganglionectomy and induction of coarctation hypertension. Successful denervation was demonstrated by a shift of the norepinephrine dose-response curve to the left in the denervated ears as well as a decreased threshold (p less than 0.05), an increased slope (p less than 0.02) and a decreased M50 (p less than 0.01) (the concentration of norepinephrine that produced 50% of the maximal vasoconstrictor response to norepinephrine). The flow-pressure curves of the denervated compared to the innervated ears at maximal dilation (used as a functional assessment of structural alterations) did not differ. The maximal pressor responses to vasopressin and barium chloride (used as an indication of smooth muscle mass) also did not differ. These results suggest that sympathetic innervation does not importantly influence blood vessel structure during coarctation hypertension in contrast to the changes we previously found in normotensive rabbits.

Spontaneously hypertensive rats (SHR) have been shown to have an increased capacity for superior cervical sympathetic nerve activity which may protect against stroke (Mueller et al: Stroke 13: 115, 1982). Sympathetic nerve activity has never been examined in the stroke-prone substrain of SHR (SP). In this study we measured superior cervical sympathetic nerve activity during rest and during a maximal sympathetic response in SHR, SP, and their normotensive controls, Wistar-Kyoto (WKY). The resting superior cervical sympathetic nerve activity of SP was significantly less than SHR (p less than 0.02) but not different from WKY. During central ischemia, used to induce maximal sympathetic response, the increase in SP sympathetic nerve activity was significantly less than SHR (p less than 0.001) but was not different from WKY. This diminished capacity for elevated superior cervical sympathetic nerve activity in stroke-prone SHR may relate to their increased predisposition to stroke because sympathetic hyperactivity cannot protect cerebral vessels during acute hypertension.

Regulation of bile acid synthesis in man is incompletely understood, in part because of difficulty in making measurements over short time periods when the enterohepatic circulation is intact. We investigated the possibility of a diurnal rhythm of bile acid synthesis in three human subjects given [26-14C]cholesterol. When this isotope of cholesterol, which is randomly labeled in the 26 and 27 positions, is converted to bile acid, the 14C is released as propionic acid randomly labeled in the 1 and 3 positions. The labeled propionic acid is then oxidized to 14CO2, output of which is a function of bile acid synthesis. However, delays in transit of the 14C through propionic acid and CO2-HCO-3 pools would shift the phase and dampen the amplitude of 14CO2 output relative to an existing diurnal rhythm of bile acid synthesis. Therefore, using constant infusion methods, we determined the turnover constants for conversion to 14CO2 of [1-14C]propionic acid and [3-14C]propionic acid to be 0.36-0.59 h-1 and 0.14-0.16 h-1, respectively. Using these constants and modeling the diurnal rhythm as a cosine function, we determined that amplitude of 14CO2 output from [26-14C]cholesterol was reduced 35% and acrophase was delayed 2.4-3.0 h relative to the diurnal rhythm of bile acid synthesis. None of the diurnal rhythm in 14CO2 output from [26-14C]cholesterol resulted from diurnal variation in propionic acid or CO2-HCO-3 metabolism since constant infusion of [1-14C]propionic acid and [3-14C]propionic acid for 30 h revealed no diurnal variation in output of 14CO2. These studies demonstrate for the first time that humans with an intact enterohepatic circulation have a diurnal rhythm of bile acid synthesis with an amplitude of +/- 35-55% around mean synthesis, and an acrophase at about 9 a.m.

Phenylpropanolamine (PPA) is a drug that has been associated with serious side effects including stroke. It is often combined with caffeine in diet preparations and "look-alike" pills. In order to determine if PPA/caffeine can lead to stroke in normotensive and/or hypertensive rats, we administered the combination in six times the allowed human dose calculated on a per weight basis for the rats two times per day for five days. Subarachnoid and cerebral hemorrhage was noted in 18% of the hypertensive rats. A single PPA/caffeine administration (same dose) lead to acute hypertension in both the normotensive and hypertensive animals. These results suggest that PPA/caffeine can lead to cerebral hemorrhage in previously hypertensive animals when administered in greater than the allowed dosage. An acute elevation in blood pressure may be a contributing factor.

Nine achondroplastic dwafts were studied with computerized tomography, skull and cervical spine films, and psychometric testing. All had large ventricles that ranged from the top limits of normal to severe hydrocephalus. Three had enlarged cortical sulci. Skull and cervical spine films were typical for achondroplasia, but in addition significant asymmetry of the petrous ridge was noted. Psychometric testing disclosed generally average intelligence quotients which fell below the expected performance level in each case. These findings may be explained on the basis of the abnormal endochondral bone formation found in achondroplasia.

The purpose of this study was to determine resting and maximal superior cervical sympathetic nerve activity in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) at five and ten weeks of age as hypertension was developing. Basal cervical sympathetic nerve activity (SNA) of five week SHR was 58 +/- 3 muv* which was significantly elevated over age-matched WKY (SNA = 30 +/- 4 muv, *p less than 0.001) and ten week SHR (SNA = 30 +/- 4 muv, *p less than 0.001) as well as ten week WKY (SNA = 24 +/- 4 muv, *p less than 0.001). Thus, during basal conditions five week SHR nerve traffic was approximately two times that found in age-matched WKY as well as in ten week SHR and WKY. The peak sympathetic nerve activity in response to rapid hemorrhage in five week SHR (215 +/- 16 muv*) was significantly elevated over the maximal response of WKY (140 +/- 23 muv) (*p less than 0.02). Ten week SHR also reached a maximal sympathetic nerve activity (187 +/- 28 muv*) that was significantly elevated over WKY (100 +/- 15 muv) (*p less than 0.02). Thus, both five and ten week SHR had a greater capacity for elevated nerve activity following rapid hemorrhage than age-matched WKY. The elevation in resting cervical sympathetic activity in five week SHR, and the elevated capacity for sympathetic neural response in both five as well as ten week SHR, are consistent with a central nervous system abnormality in SHR that could relate to the previously described protective influence of sympathetic nerves on SHR cerebral blood vessels as hypertension is developing.

The purpose of this study was to determine the effect of chronic sympathetic denervation on cerebral vessels of the spontaneously hypertensive rat when superior cervical ganglionectomy was performed in adulthood. In a previous study, we have demonstrated increased protein transfer across the cerebral vessels of the chronically denervated hemisphere when superior cervical ganglionectomy was performed in adolescent spontaneously hypertensive rats. After four weeks of sympathetic denervation, the adult rats in this study did not demonstrate increased protein transfer in the denervated compared to the innervated cerebral hemisphere. Thus, the "trophic" effect of sympathetic nerves on cerebral vessels appears to be age-related.

Vascular angiotensin converting enzyme could contribute to the elevated vascular resistance found in hypertension. The purpose of this study was to determine if angiotensin converting enzyme activity was present in the hindquarter vasculature of one model of hypertension, the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY). We evaluated the effect of a maximal blocking dose of captopril (0.5 mg) on the angiotensin I pressor response during the infusion of the hindquarter with an artificial perfusate. Angiotensin I (1000 ng/ml) produced a significant increase in peripheral vascular resistance (PVR) in both SHR and WKY, but the increase was greater in SHR. Captopril inhibited the elevation in PVR in both. A lower concentration of angiotensin I (250 ng/ml) produced a significant and similar pressor response in SHR (less than the pressor response to 1000 ng/ml) and WKY (same as the pressor response to 1000 ng/ml). Again, captopril prevented the elevation in PVR to A1 in both SHR and WKY. Because these studies were performed using an artificial perfusate, angiotensin converting enzyme must be present in the SHR and WKY hindquarter vasculature including resistance vessels.

The purpose of this study was to determine the effect of activation of sympathetic pathways during seizures on cerebral blood flow and integrity of the blood-brain barrier. We measured cerebral blood flow with microspheres and disruption of the blood-brain barrier with labeled albumin in cats. One cerebral hemisphere was denervated by cutting the superior cervical sympathetic trunk on one side. During bicuculline-induced seizures, superior cervical sympathetic nerve activity increased about threefold. Blood flow to the innervated hemibrain was significantly lower than flow to denervated hemibrain. However, in relation to the total increase in flow, this effect of nerves was minor. Blood-brain barrier permeability increased about sixfold during seizures, but there was no difference between the innervated and denervated sides of the brain. We conclude that sympathetic nerves attenuate the increase in cerebral blood flow during seizures, despite the increase in metabolism, but this effect is small. Activation of sympathetic nerves does not reduce disruption of the blood-brain barrier during seizures.

We used a noninvasive technique--the directional Doppler examination--to determine the direction of flow in an emissary vein of achondroplastic dwarfs. Normally, the direction of flow in this emissary vein is into the orbit. The direction of flow in all 10 achondroplastic dwarfs whom we studied was away from the orbit. This indicates that ophthalmic vein blood flow is reversed in achondroplastic dwarfs, perhaps because there is a pressure gradient between the intracranial and extracranial venous systems.

Disruption of the blood-brain barrier (BBB) during acute hypertension may contribute to hypertensive encephalopathy. In this study we tested the hypothesis that, in chronic hypertension, vascular changes might influence the susceptibility of the BBB to disruption. Spontaneously hypertensive rats (SHR) and normotensive rats (WKY), 3-4 months of age, were anesthetized and acute hypertension was produced by infusing phenylephrine intravenously (i.v.). Permeability of the BBB was studied with radioactive iodine serum albumin (RISA) injected i.v. The ratio of brain-to-blood RISA was used as an index of permeability of the BBB expressed as protein transfer. In both SHR and WKY at resting arterial pressure, the protein transfer was less than 0.10%. In WKY exposed to acute hypertension (mean arterial pressure increased by 87 +/- 7 mm Hg), the protein transfer was 2.77 +/- 0.60%. In SHR with acute hypertension superimposed on chronic hypertension (arterial pressure increased by 80 +/- 7 mm Hg), the protein transfer was 1.16 +/- 0.45% (p less than 0.05, SHR vs WKY). These data suggest that cerebral vessels are less susceptible to disruption of the BBB by acute hypertension in SHR than in WKY. We speculate that the finding of reduced susceptibility to BBB disruption in chronic hypertension may explain, in part, the apparent susceptibility of previously normotensive patients to acute hypertensive encephalopathy.

It has been shown that the blood-brain barrier (BBB) of chronically hypertensive adult spontaneously hypertensive rats (SHR) is less susceptible to disruption during acute superimposed hypertension than normotensive controls. The purpose of this study was to determine if the BBB of young SHR, not yet markedly hypertensive, was similarly protected during super-imposed acute hypertension. Spontaneously hypertensive rats (n = 22) and normotensive Wistar-Kyoto rats (WKY) (n = 23) 4-5 weeks of age were anesthetized with secobarbital sodium (50 mg/kg) intraperitoneally and acute hypertension was produced by an intravenous injection of norepinephrine (75 micrograms). Permeability of the BBB was studied with radioactive iodine serum albumin (RISA) injected intravenously. The ratio of brain-to-blood RISA X 100 was used as an index of permeability of the BBB expressed as % protein transfer. In WKY exposed to acute hypertension mean arterial pressure increased by 52 +/- 2 mmHg and in SHR the increase was 49 +/- 3 mmHg. The protein transfer of the cerebral hemispheres was 1.17 +/- 0.30% in WKY and 0.90 +/- 0.20% in SHR (P less than 0.40). These data indicate that BBB protein transfer during acute superimposed hypertension does not differ between young SHR and WKY. Thus, the reduced susceptibility to BBB disruption in chronically hypertensive adult SHR is not present in young SHR, making them as susceptible as WKY to cerebral complications related to protein transfer during acute hypertension.