Mitochondrial cytochrome c release: a factor to consider in mitochondrial disease?

The pathogenesis of mitochondrial disorders has largely focused on the impairment of cellular energy metabolism. However, mitochondrial dysfunction has also been implicated as a factor in the initiation of apoptosis due to the translocation of cytochrome c, from mitochondria to the cytosol, and the subsequent cleavage of pro-caspase 3. In this study, we determined the cytochrome c content of cytosols (skeletal muscle) prepared from 22 patients with evidence of compromised mitochondrial electron transport chain enzyme activity and 26 disease controls. The cytochrome c content of the mitochondrial electron transport chain-deficient group was found to be significantly (p < 0.02) elevated when compared with the control group (63.7 +/- 15.5 versus 27.7 +/- 2.5 ng/mg protein). Furthermore, a relationship between the cytosolic cytochrome c content of skeletal muscle and complex I and complex IV activities was demonstrated. Such data raise the possibility that mitochondrial cytochrome c release may be a feature of mitochondrial disorders, particularly for those patients with marked deficiencies of respiratory chain enzymes. Whether initiation of apoptosis occurs as a direct consequence of this cytochrome c release has not been fully evaluated here. However, for one patient with the greatest documented cytosolic cytochrome c content, caspase 3 could be demonstrated in the cytosolic preparation. Further work is required in order to establish whether a relationship also exists between caspase 3 formation and the magnitude of respiratory chain deficiency.

A portable energy-sensitive cosmic neutron detection instrument.

The construction and testing of a portable energy-sensitive neutron instrument are described. This instrument has been designed and constructed for the primary purpose of characterizing cosmic-ray neutron fields in the upper atmosphere and in cosmic reference field facilities. The instrument comprises a helium-3 proportional counter surrounded by 15 mm of lead and 140 mm of polyethylene creating a spherical structure with a diameter of 34 cm. The instrument also incorporates 12 boron-coated diodes, six on the outside of the polyethylene layer with six placed within the structure. The dimensions, materials, and arrangement of these in the instrument have previously been optimized with the MCNPX Monte Carlo simulation software to provide a compromise between the requirements of portability and spectral response. Testing took place at several locations and experimental data from the instrument's operation at the high-altitude Jungfraujoch laboratory in the Swiss alps are presented.

Ocular inflammation stimulated by intravitreal interleukin-8 and interleukin-1.

Interleukin-8 (IL-8), a cytokine with neutrophil chemotactic and activating properties, is known to be stimulated by IL-1. Fischer rats are more resistant to inflammation than Lewis rats probably due to a higher corticosteroid stress response. To determine the role of IL-8 in ocular inflammation, the effect of intravitreal injection of IL-8 was compared with that of IL-1 in both Lewis and Fischer rats. The IL-8, IL-1 alpha, or sterile balanced salt solution (control) was injected into one eye of each animal. Both IL-8 and IL-1 alpha caused inflammation in the eye of both strains, as detected by leukocyte counts of the anterior chamber and histopathologic examination. The eyes of animals injected with a cytokine had significantly higher numbers of leukocytes compared with eyes of control animals. Histopathologic examination confirmed these findings. The IL-1 alpha induced inflammation more consistently and more severely than the most effective dose of IL-8. This finding agreed with the concept of IL-1 initiating a cascade of inflammatory mediators including IL-8, which acts more specifically on a smaller population of leukocytes. A contralateral response was observed in the uninjected eye of experimental and control animals. The contralateral response in animals receiving the cytokines was significantly greater than that in controls. Lewis rats show a higher inflammatory response to the injections than do the Fischer rats. These data suggest that IL-8 may be active as one component in neutrophil-mediated ocular inflammation.

A clinical evaluation of a radiopaque material in the restoration of anterior and posterior teeth.

The clinical performances of Adaptic and Radiopaque Adaptic restorative materials are compared according to their properties of retention, marginal integrity, and color change. The advantage of radiopacity in Class I and Class IV restorations of anterior and posterior teeth is discussed.

Clinical comparison of sealant and bonding systems in the restoration of fractured anterior teeth.

This study compared the clinical performance of the ultraviolet light-initiated fissure sealant and composite system (Nuva-Seal/Nuva-Fil) with a chemically activated system (Adaptic Bonding Agent/Adaptic). A total of 63 fractured permanent anterior teeth involving only enamel and dentin were restored. All teeth were isolated with rubber dams, and the acid etch technique was used. The degree of success was determined from an assessment of retention, marginal integrity, marginal staining, and color change. The restorations were evaluated at 3, 6, 12, 18, and 24 months postoperatively. Of the restorations, 93% were retained in both groups of patients after two years. Varying degrees of marginal problems (lack of marginal integrity or marginal staining) were directly attributable to the extension of the bonding medium and the composite in relation to the conditioned enamel and to each other. Repolishing of the margins should be considered at the time of the recall appointments to improve the quality of restoration.

BIO-SPEAD: a parallel computing environment to accelerate development of biologic signal processing algorithms.

We have created BIO-SPEAD (pronounced speed), a BIOlogical Signal Processing Environment for Algorithm Development. BIO-SPEAD is designed to accelerate development of complex algorithms which integrate information derived from single or multiple physiologic waveforms. BIO-SPEAD currently performs all of the basic analyses of several arterial blood pressure waveforms, and allows the user to utilize the results of those low-level analyses for development of more complex algorithms. We utilized a parallel programming architecture called the Process Trellis which keeps the different tasks, or processes, within BIO-SPEAD independent of each other. Additionally, we have developed a graphics interface to enable the user to visualize the waveform under analysis, the low-level system analysis, and the internal workings of the algorithm under development. The system has been used for several algorithm development projects and has demonstrated its utility.

Early infancy oral health care.

Parents need guidance regarding the oral health of their infant children. The dental profession is best suited by training and experience to provide this information. Issues such as timing of the first appointment, examination techniques, caries prevention, fluoride management and non-nutritive sucking habits are discussed. Counseling of parents can minimize many of the technical interventions necessary in infancy and later childhood.

Electrostatic mode excitation in electron holes due to wave bounce resonances.

A kinetic theory of resonant interaction between electrostatic waves and the bounce motion of electrostatically trapped electrons is developed. Precise criteria are derived for the stability of electrostatic potential structures which trap electrons in a highly magnetized plasma. The theory explains the energy transfer from electron phase space holes to waves observed in simulations. It may also account for the destabilization of electrostatic waves propagating obliquely to the geomagnetic field and some characteristics of the holes as observed in the auroral ionosphere.

An innovative dicrotic notch detection algorithm which combines rule-based logic with digital signal processing techniques.

Automated, real-time localization of the dicrotic notch, a component of the arterial pressure waveform, represents a deceptively complex problem in computerized biomedical signal processing. The high-frequency nature of the notch can make it difficult to distinguish from artifactual noise or from other high-frequency physiological components of the waveform. In addition, the contour of the notch varies with vascular status and with propagation through arterial beds, requiring any detection algorithm to recognize various possible notch conformations. Finally, location of the notch along the waveform may vary widely depending on other hemodynamic variables, further complicating detection algorithms. We have reviewed various published algorithms and have implemented a number of them to determine the strengths and shortcomings of each. We then developed a reliable and accurate hybrid algorithm which utilizes the strengths of the various algorithmic approaches reviewed; after analyzing the waveform, the algorithm selects the most appropriate method for accurate notch localization based on a series of waveform features. The application of rule-based logic represents a relatively unique approach to digital signal processing.

Interleukin-2: solid-tumor therapy.

Interleukin-2 (IL-2) is a soluble factor produced by T cells that stimulates growth and activity of lymphocytes and other immune cells. First noted in murine studies, the antitumor efficacy of IL-2 has been shown to induce partial and complete regression of some tumors in human clinical trials over the past decade. Although the initial clinical success of IL-2 was in combination with lymphokine-activated killer cells, IL-2 alone has subsequently been shown to be equally efficacious. Combinations of cytokines and chemotherapies with IL-2 have been generally inconclusive and disappointing with the possible exception of interferon-alpha. Toxicities of IL-2 are common and often dose limiting. Symptomatic therapy has allowed patients to tolerate somewhat higher doses, but has not addressed the underlying mechanisms of these toxicities which may involve mediators such as tumor necrosis factor, interferon-gamma, and nitric oxide. Clinical studies assessing these factors for their involvement in the antitumor effects of IL-2 as well as its toxicities may allow better understanding of IL-2, and perhaps lead to improved cancer therapies.

A parallel computing interface to facilitate development of physiologic signal processing algorithms.

We have created a system to aid in the development of algorithms related to the blood pressure waveform. The system performs all of the basic analyses of the waveform, and allows the user to utilize the results of those analyses for the algorithm under development. We have used a parallel programming architecture which keeps the different tasks, or processes, within the system independent of each other. Additionally, we have developed a graphics interface to enable the user to visualize the waveform, the system analysis, and the internal workings of the algorithm under development.

Cytotoxicity of human serum for Leishmania donovani amastigotes: antibody facilitation of alternate complement pathway-mediated killing.

Mechanisms that mediate recovery from leishmanial infection have not been fully characterized but are generally believed to involve interactions between T lymphocytes and macrophages. A major role for serum-mediated effector mechanisms in the protection of humans from reinfection with Leishmania, however, has not been ruled out. In this report, amastigotes of L. donovani were incubated with dilutions of serum from normal subjects and from patients with kala-azar. Normal serum was cytotoxic for parasites at a dilution of greater than or equal to 1:20. Cytotoxicity did not occur in the presence of EDTA, was abolished by heating serum to 56 degrees C for 30 min, and was not diminished by prior adsorption of normal serum with parasites at 0 degree C. Killing proceeded normally in the presence of magnesium-ethylene glycol-bis(beta-aminoethyl ether)-N, N-tetraacetic acid, however, and was fully effected by C2-deficient serum. These studies indicated that killing of amastigotes, unlike that of promastigotes, was mediated via the alternate pathway of serum complement. In further studies, cytotoxicity of normal serum was enhanced three- to fivefold by factors in patient serum. This enhanced cytotoxicity also proceeded via the alternate complement pathway. Factors that enhanced cytotoxicity were characterized as parasite-specific immunoglobulin G: they eluted with immunoglobulin G on column chromatography, were adsorbed by immobilized staphylococcal protein A, and were not removed from the parasite surface by extensive washing. Thus, infection of individuals with L. donovani resulted in the production of a new, qualitatively and quantitatively distinct immune mechanism directed against the amastigote form of the parasite, namely, antibody-directed, alternate complement pathway-mediated cytotoxicity. These results provide a mechanistic framework for a role of humoral factors in human resistance to reinfection with L. donovani.

Design of a clinical alert system to facilitate development, testing, maintenance, and user-specific notification.

Creation and maintenance of electronic clinical alerts within a hospital's electronic medical record (EMR) or database poses a number of challenges. Development can require significant programming effort. Final testing should ideally be performed in a real clinical environment without clinician notification, which may create technical challenges. After an alert is in production, modifications may become necessary in response clinician feedback, changes in clinical factors, or technical issues. Changes may be required in the knowledge base utilized by the alert or in the presentation of the alert condition to the clinicians. Occasionally, different users within the clinical environment may wish to have the same alert data presented differently. We have developed a strategy which allows development of multi-functional alerts and facilitates modification of alert function and/or presentation with minimal to no programming effort. Some elements of this scheme may be appropriate for incorporation into clinical alerting standards.

Addition of interleukin 12 to low dose interleukin 2 treatment improves antitumor efficacy in vivo.

Interleukin 12 (IL-12) enhances lysis mediated by NK- and lymphokine activated killer (LAK) cells. It also causes proliferation of IL-2 stimulated T and NK cells in vitro. For these IL-2 complementing properties murine pulmonary metastases of a coloncarcinoma line were treated with IL-12 and IL-2 or with the individual agents. Results were compared to sham treated controls. IL-2 alone mediated significant tumor reduction but provoked pulmonary edema and concomittand toxicity, graded in three steps. IL-12 combined with an IL-2 dose reduced by 81% still resulted in significant antitumoral activity. Toxicity, however, was not discernable from sham treated controls. IL-12 thus appears as an attractive cytokine for combination with IL-2 in antitumor therapy. Particularly treatment of tumors, like gastrointestinal tract cancers, so far mainly resistant to cell mediated antitumor therapy, might profit from this approach.

Inhibition of nitric oxide synthesis does not improve interleukin-2-mediated antitumor effects in vivo.

Nitric oxide (NO) decreases cytotoxicity and proliferation of cytotoxic lymphocytes (CTLs) in vitro. Both can be prevented by inhibitors of the NO synthase (NOS). To elucidate whether inhibition of the IL-2-induced NOS could boost efficacy of IL-2-stimulated CTLs in vivo, we assessed lung metastases in mice injected with IL-2, the NOS inhibitor aminoguanidine (AG), their combination and the diluent. No improvement was observed for IL-2 + AG compared to IL-2 while NO production was normalized. Since NO causes one of the two major side effects of IL-2 treatment, hypotension, we further studied whether capillary leak could be attributed to NO, too. While IL-2-inducible NO was reduced to control levels by AG, pulmonary edema was unaffected. Thus a decrease in NO does not improve antitumor effects of IL-2-stimulated CTLs nor does it attenuate IL-2-associated capillary leak.