Good Samaritan University Hospital

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Assessment of standing balance is essential to the treatment of instability in the neurologic patient. The development of clinical techniques for evaluating instability is dependent on a thorough understanding of sensory and motor processes underlying normal balance control. Motor processes in balance control coordinate the action of trunk and leg muscles into discrete synergies that minimize sway and maintain the body's center of mass within its base of support.1,2 Sensory processes in balance control involve interaction among orientation inputs from somatosensory (proprioceptive, cutaneous, and joint), visual, and vestibular systems. Despite the availability of multiple sensory inputs, the central nervous system generally relies on only one sense at a time for orientation information.3 For healthy adults, the preferred sensory input for the control of balance is somatosensory information from the feet in contact with the support surface.

Patients whose deficits were limited to clinically well qualified vestibular disorders have been exposed to a number of altered support surface and visual environments while standing unsupported. A six- degrees-of-freedom platform employing movable support surfaces for each foot and a movable visual surround deprived patients of normal inputs derived from a fixed level support surface and from an immobile surround. Various tests employing EMG, force, and body movement recording identified quantitative changes in the patients' strategy for the relative weighting of proprioceptive, vestibular, and visual inputs. The most dramatic performance deficit of patients was their inability to suppress the influence of visual and proprioceptive inputs appropriately whenever motions of external surface disturbed the orientation information provided by these inputs. Thus, the more mildly afflicted patients experienced instability not so much because of the loss of vestibular inputs directly to posture but because of their inappropriate responses to proprioceptive inputs and vision. Discussion is centered on the role of vestibular input as an internal reference system for orientation about which adaptive changes in proprioceptive and visual inputs are made.

The following study examined two aspects of balance control in the older adult: the coordination of the timing and the amplitude of muscle responses to postural perturbations, and the ability of the participant to reorganize sensory inputs and subsequently modify postural responses as a consequence of changing environmental conditions. Coordination of muscle activity in postural responses of twelve elderly (sixty-one to seventy-eight years) participants were compared to those of young (nineteen to thirty-eight years) adults using a movable platform and recording the electromyographic activity of muscles of the legs. The following changes were noted in the timing and amplitude of muscle activity within a postural response synergy: increases in the absolute latency of distal muscle responses were observed in all older adults; in five of the twelve older adults temporal reversals of proximal and distal muscle response onset were observed; and there was a breakdown in the correlation of the amplitude of responses within a synergy. The ability of the older adult to balance under conditions of reduced or conflicting sensory information was also impaired. When confronted with functionally inappropriate visual and/or somatosensory inputs, half of the older group lost balance. In most instances, however, the older participants were able to maintain stability during subsequent responses to conflicting stimuli.

Recent studies have suggested that rituximab has clinical activity and modulates antiapoptotic proteins associated with drug resistance in chronic lymphocytic leukemia (CLL). We performed a randomized phase 2 study to determine the efficacy, safety, and optimal administration schedule of rituximab with fludarabine in previously untreated CLL patients. Patients were randomized to receive either 6 monthly courses of fludarabine concurrently with rituximab followed 2 months later by 4 weekly doses of rituximab for consolidation therapy or sequential fludarabine alone followed 2 months later by rituximab consolidation therapy. A total of 104 patients were randomized to the concurrent (n = 51) and sequential (n = 53) regimens. During the induction portion of treatment, patients receiving the concurrent regimen experienced more grade 3 or 4 neutropenia (74% versus 41%) and grade 3 or 4 infusion-related toxicity (20% versus 0%) as compared with the sequential arm. The consolidation rituximab therapy was tolerated well in both arms. All other toxicities were similar in the 2 arms. The overall response rate with the concurrent regimen was 90% (47% complete response [CR], 43% partial response [PR]; 95% confidence interval [CI], 0.82-0.98) compared with 77% (28% CR, 49% PR; 95% CI, 0.66-0.99) with the sequential regimen. With a median follow-up time of 23 months, the median response duration and survival have not been reached for either regimen. Rituximab administered concurrently with fludarabine in previously untreated CLL patients demonstrates marked clinical efficacy and acceptable toxicity. Phase 3 studies using this combination approach for patients with CLL are warranted.

Age- and pathology-related changes in the relative contributions of visual and somatosensory inputs to dynamic balance control were evaluated. Young adults (mean age = 25, SD = 4) were compared to older adults (mean age = 68, SD = 5). Electromyographic responses were collected when subjects' balance was perturbed on a movable platform. The amounts of visual information and of somatosensory input at the ankle were manipulated. Muscle response latencies, losses of balance, and muscle sequencing were analyzed. Muscle response latencies did not differ across age groups. Loss of balance data indicated that older adults were less stable under conditions in which peripheral vision was occluded and ankle somatosensation was limited (only foveal vision and vestibular input remaining). Older adults showed more antagonist muscle activation and used muscle sequences not seen in young adults (e.g., hip strategy). These effects were exaggerated among subjects in whom borderline pathology had been diagnosed.

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

Although considerable progress has been made in elucidating the molecular events occurring during kinin generation by both the plasma kinin-forming system and the tissue kallikrein system, it is only in recent years that we have come to appreciate their potential role in inflammation in a wide variety of diseases. The importance of the tissue kallikrein system depends upon secretion of the active form of the requisite enzyme in the presence of a source of kininogen. Since tissue kallikreins are widely distributed in tissues, and since lymph and interstitial fluid contains kininogen (271), a local milieu for potential kinin formation is always present. The plasma system will be activated secondary to inflammation initiated by some other process. There may be endothelial or epithelial damage exposing connective tissue. Plasma leakage caused by release of some other permeability factor (including kinin made by tissue kallikrein) would thus lead to activation of the plasma cascade in many forms of inflammation. As with all mediators, however, the contribution of kinins to an inflammatory response can only be definitively evaluated if their actions can be selectively antagonized. Competitive receptor antagonists have recently been synthesized (228) and will, we hope, soon be available for administration to humans. Should these compounds prove effective in vivo, they could be used in conjunction with currently available assays for kallikreins, kininogens, kinins, and their various inactivated or degraded products, to provide new insights into the role of these systems in the pathogeneses of inflammatory diseases.

Normal young children ranging in age from 1 1/2 to 10 years were assessed in a number of experimental paradigms testing the ability to adapt quickly their strategy of control to altered support surface and visual conditions. The experimental protocols, using a movable platform and visual surround, and the analytic techniques, using EMGs and measures of reaction forces and body motions, were identical to those employed in a complementary study in this issue (Nashner, L. M., F. O. Black, and C. Wall, III (1982) J. Neurosci. 2: 536-544). The structure of automatic postural adjustments in young children was, with the exception of greater variability, similar to that of adult subjects studied previously. However, young children below the age of 7 1/2 years were unable to suppress systematically the influence of inputs derived from the support surface or from vision when these provided inappropriate orientation information due to the motion of these surfaces. The discussion emphasizes that the automatic postural adjustments and the context-dependent reweighting of support surface, vestibular, and visual inputs are organizationally separate processes and that the hierarchically lower level automatic process matures before the higher level adaptive processes.

In the course of a controlled study to evaluate different forms of immunotherapy for subjects with insect-sting hypersensitivity, we observed 11 subjects who had systemic cutaneous urticarial reactions and 3 subjects who experienced systemic anaphylaxis. With the exception of tachycardia, there were no cardiopulmonary changes in the subjects with urticaria, whereas the major manifestation of anaphylactic shock in the other three subjects was severe hypotension that was probably secondary to peripheral vasodilation. Significant abnormalities in gas exchange developed in two subjects. In one, bronchospasm precipitated a respiratory arrest followed by endotracheal intubation with mechanical ventilation. Although plasma histamine levels were not related to the development of cutaneous reactions, the plasma histamine levels correlated with the severity and duration of the cardiopulmonary changes observed during anaphylactic shock. The two subjects with the most severe shock showed evidence of intravascular coagulation characterized by a diminution of Factor V, Factor VIII, fibrinogen, and high molecular weight kininogen, as well as changes in components of the complement system. Standard therapy with epinephrine and fluids, usually recommended for the treatment of systemic anaphylaxis, did not immediately reverse either the hemodynamic or the respiratory abnormalities in the two subjects with the most severe anaphylactic shock. Hemodynamic recovery was gradual and did not seem directly related to any specific therapeutic intervention.

Abstract The hemolysis of sheep red blood cells (SRBC) occurs via the classical complement pathway and is blocked by ethylene glycol bis-amino tetraacetate (EGTA). By contrast, fresh normal human serum in EGTA buffer was found to cause >90% hemolysis of unsensitized rabbit red blood cells (RaRBC) at a final dilution of 1:15. Absorbing human serum with RaRBC at 0°C will remove only 45% of this hemolytic activity and the same activity is present in human hypogammaglobulinemic serum. When rabbit lymphocytes were incubated with human serum in EGTA buffer, complement fixation occurred on their surface which was demonstrated with radiolabeled antibodies to human C3 or as “blocking” of the complement receptor. With purified complement components it was shown that the EGTA buffer completely blocked C1 but not C4, C2, or the late complement components. These findings show that a rabbit cell surface component can activate the alternate pathway of complement in human serum and suggest that this activation does not involve antibody.

Specific IgE anti-ragweed antibodies (IgEAR) were measured over two years in two groups of highly sensitive patients treated (immunized) with either ragweed extract or placebo and in a third group of placebo-treated, relatively insensitive patients. The IgEAR on the patients' basophils were assessed by ragweed antigen E (AgE)-induced histamine release; blocking (IgG) antibodies were measured by their ability to inhibit AgE induced histamine release. These data were evaluated against the clinical severity of ragweed hay fever in each patient. WE FOUND THAT: (a) In placebo treated patients IgEAR usually declined gradually prior to the ragweed season and were boosted by environmental exposure to ragweed pollen. (b) In immunized patients the IgEAR rose at the beginning of treatment, but fell as immunotherapy proceeded; by the end of the second year the levels had decreased in 18/19 patients. (c) The increase in blocking antibody during immunotherapy correlated significantly (P < 0.05) with the decrease in serum IgEAR. (d) Judged by their sensitivity to AgE induced histamine release, IgEAR on basophils correlated significantly with IgEAR in the serum of untreated patients (P < 0.01). (e) The highly sensitive placebo treated patients' symptom scores were significantly correlated with their IgEAR in serum (P < 0.01) and with the sensitivity of their basophils to AgE-induced histamine release (P < 0.01). Neither correlation was observed in the relatively insensitive patients. (f) In the treated group the IgEAR measurements predicted neither the degree of their illness nor their clinical improvement We conclude tha IgE antibody measurements may be useful in the assessment of the severity of reaginic allergy in highly sensitive patients. Its use in modestly sensitive patients requires patients requires further study, as does the inverse association between IgE and IgG antiragweed antibodies.

The quantitative somatosensory thermotest (QST) assesses the function of afferent channels concerned with sensory submodalities served by small calibre fibres. Measured ramps of ascending or descending temperature are applied to the skin through a Peltier contact thermode, and detection thresholds are recorded as the subject signals the onset of a particular sensation. The present study describes underlying principles, methodological aspects and normal reference values for the QST. In patients, measurement of thresholds for cold sensation, warm sensation, cold-induced pain and heat-induced pain, applied to 465 individuals, yielded 13 abnormal patterns segregated into three main groups: (i) thermal (cold or warm) hypoaesthesia; (ii) thermal hyperalgesia (abnormally reduced threshold for cold and/or heat induced pain); (iii) thermal hypoaesthesia combined with thermal hyperalgesia. Critical analysis of these results yielded a number of observations of general relevance: (i) thermal specific (warm or cold) hypoaesthesia and thermal (heat or cold) hyperalgesia may occur in the absence of hypoaesthesia for tactile submodalities served by large calibre afferents; (ii) cold hypoaesthesia and warm hypoaesthesia may dissociate from each other; (iii) thermal pain hyperalgesias may occur in the absence of hypoaesthesias for specific cold or warm sensations; (iv) cold hyperalgesia and heat hyperalgesia may dissociate from each other. Thus, a negative routine sensory examination and unimpaired sensory nerve action potentials do not exclude possible somatosensory dysfunction. Furthermore, while most methods of sensory testing only document normality or deficit, the QST permits additional documentation of hyperalgesia, a positive sensory phenomenon that implies unusual pathophysiologies such as sensitization of receptors, central hyperexcitability, disinhibition or, possibly, ectopic nerve impulse discharge. This psychophysical test does not specify the level within afferent channels, between skin and brainmind, where the abnormality resides. It is recommended that the QST for all four thermal specific and thermal pain functions be incorporated in routine neurological assessment.

Using a recently developed model of nasal challenge, we have obtained data that clearly demonstrate, for the first time, kinin generation during a local allergic reaction in vivo. Allergic individuals (n = 8) and matched nonallergic controls (n = 8) were challenged intranasally with the appropriate antigen and nasal washes were taken before and after challenge. Washes were assayed for kinin, histamine, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity. Increased kinin generation was found by radioimmunoassay (RIA) in the nasal washes of all the allergics (5,560 +/- 1,670 pg/ml) but in none of the controls (38 +/- 16 pg/ml). The presence of kinin was highly correlated with that of histamine and TAME-esterase activity and with the onset of clinical symptoms (P less than 0.001). Serial dilutions of nasal washes produced RIA displacement curves that paralleled the standard curve, and recovery of standard kinins that were added to nasal washes was 100 +/- 4% (n = 14). Kinin recovery was identical in both allergics and controls and did not vary significantly with antigen challenge. The immunoreactive kinin in nasal washes was stable to boiling and not precipitated by ethanol, but completely destroyed by carboxypeptidase B. It was evenly distributed between the sol and gel phases of nasal washes. High performance liquid chromatography analysis of the immunoreactive kinin in nasal washes showed it to be a mixture of lysylbradykinin and bradykinin. We conclude that kinins are produced during local allergic reactions in the nose and may contribute to the symptomatology of the allergic response.

Loss of independent eating capacity is a major problem for the institutionalized elderly. Few studies have examined the factors associated with loss of functional eating capacity. The authors cross-sectionally studied 240 residents of a skilled nursing facility, classified their functional eating status, identified correlated deficits, and followed these residents for six months. Information was gathered through questionnaires, chart review, and physical examinations. Residents were stratified into independent (68%, N = 164) and dependent (32%, N = 76) eating status groups according to the need for physical assistance during meals. Dependency status did not correlate with age (P = .88) or weight loss (P = .27). Loss of independence in eating was associated with impaired mobility (P = .0001), impaired cognition (P = .0001), modified consistency diets (P = .0001), upper extremity dysfunction (P = .0001), abnormal oral-motor examinations (P = .0002), absence of teeth and dentures (P = .002), behavioral indicators of abnormal oral and pharyngeal stages of swallowing (P = .0001), and increased mortality within six months (P = .0001). Eating dependency is therefore associated with multiple impairments and early mortality.

Direct carotid-cavernous fistulas are high-flow shunts with a direct connection between the internal carotid artery and the cavernous sinus. The goals of treatment are to eliminate the fistula and preserve carotid artery patency. The authors reviewed the outcome of 98 patients with 100 consecutive direct carotid-cavernous fistulas initially treated by transarterial embolization with detachable balloons (1979-1992) at the University of Cincinnati Medical Center to evaluate the merits of this technique and to provide a standard for comparison with future treatment alternatives. Among 100 fistulas, 76 were traumatic in origin, 22 resulted from a ruptured aneurysm, and 2 were iatrogenic. The most common presentations were orbital bruit (80%), proptosis (72%), chemosis (55%), abducens palsy (49%), and conjunctival injection (44%). Eighty-eight fistulas were successfully occluded in 86 patients with detachable balloon(s), and internal carotid blood flow was preserved in 66 patients (75%). Initial attempts at balloon occlusion failed in four patients in whom the fistula eventually closed spontaneously. Five patients required direct surgery to occlude the fistula, and two were treated with nondetachable balloons; one patient died from injuries sustained from trauma. The permanent neurological complication rate was 4%, including cerebral infarction in one patient, frontal intracerebral hemorrhage in one patient, and vision loss in another patient. One death occurred related to cerebral infarction from a balloon that shifted. Transient ischemia occurred in three patients. On the basis of these results, we conclude that transarterial embolization with detachable balloons provides a high rate of fistula obliteration with low morbidity and is the best initial procedure to treat direct carotid-cavernous fistulas.

Although mediator release from mast cells and basophils plays a central role in the pathogenesis of human allergic disease, biochemical studies have been restricted to rat peritoneal mast cells and basophilic leukemia cells because they could be easily purified. We have used two new techniques of cell separation to purify human lung mast cells to 98% homogeneity. Lung cell suspensions were obtained by dispersion of chopped lung tissue with proteolytic enzymes. Mast cells were then purified from the suspensions by countercurrent centrifugal elutriation and affinity chromatography. The purified mast cells released both histamine and slow-reacting substance of anaphylaxis (SRS-A) (leukotriene C and D) during stimulation with goat anti-human IgE antibody. Moreover, these preparations were able to generate significant quantities of SRS-A (32 +/- 7 x 10(-17) LTD mole-equivalents/mast cell) at all stages of purification, indicating that a secondary cell is not necessary for the antigen-induced release of SRS.

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that target down-regulators of the anti-cancer immune response: Cytotoxic T-lymphocyte antigen-4, programmed cell death protein-1, and its ligand programmed death-ligand 1. ICIs have revolutionized the treatment of a variety of malignancies. However, many immune-related adverse events have also been described which mainly occurs as the immune system becomes less suppressed, affecting various organs including the gastrointestinal tract and causing diarrhea and colitis. The incidence of immune-mediated colitis (IMC) ranges from 1%-25% depending on the type of ICI and if used in combination. Endoscopically and histologically there is a significant overlap between IMC and inflammatory bowel disease, however more neutrophilic inflammation without chronic inflammation is usually present in IMC. Corticosteroids are recommended for grade 2 or more severe colitis while holding the immunotherapy. About one third to two thirds of patients are steroid refractory and benefit from infliximab. Recently vedolizumab has been found to be efficacious in steroid and infliximab refractory cases. While in grade 4 colitis, the immunotherapy is permanently discontinued, the decision is controversial in grade 3 colitis.

The purpose of our study was to assess the effect of cold, dry air (CDA) on the nasal mucosa of selected individuals in relation to the release of inflammatory mediators associated with mast cells. 12 subjects with a history of nasal symptoms of rhinorrhea and congestion upon cold or dry environmental exposure were challenged by nasal breathing of CDA and warm, moist air (WMA). Each subject was tested on two occasions with the order of the challenges reversed. Symptom scores were recorded, and the levels of histamine, prostaglandin (PG) D2, kinins, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity in nasal lavage fluids were measured. CDA caused a significant increase in mediator levels and in symptom scores as compared to baseline or to WMA. No significant increase in symptom scores or mediators was noted after WMA challenge, with the exception of a marginal increase in kinins. The response to CDA was similar, regardless of challenge order. Changes in mediators correlated with one another, and symptom scores correlated significantly with the levels of histamine, kinins, and PGD2. Five subjects without a history of nasal symptoms on cold air exposure had no change in mediators or symptom scores after CDA or WMA challenge. We conclude that CDA causes the release of inflammatory mediators possibly associated with mast cells and speculate that such a mechanism may be involved in the bronchospasm induced by CDA in asthmatics.