A novel lymphoproliferative/autoimmune syndrome resembling murine lpr/gld disease.

In mice, the two distinct autosomal recessive genes lpr and gld can induce a syndrome characterized by autoantibody formation and the progressive accumulation of an unusual CD4-CD8- T cell population in peripheral lymphoid tissue. This phenotype does not precisely mirror any human disease. In this report we describe two patients with a progressive lymphoproliferative disorder associated with autoimmunity. The peripheral blood and lymph nodes of these patients contained large numbers of an unusual CD4-CD8- T cell population. These CD4-CD8- T cells express surface markers characteristic of mature peripheral blood T cells (CD3, CD2, CD5), express the alpha/beta form of the T cell receptor, and do not express surface markers characteristic of immature thymocytes (CD1) or NK cells (CD16, CD56). Functionally, these cells exhibited deficient proliferation and lymphokine production upon stimulation with mitogenic antibodies to CD3 or CD2. Both proliferation and lymphokine production could be augmented by co-stimulation with an antibody directed at the CD28 determinant. The clinical and immunological features of this syndrome resemble the lymphoproliferative/autoimmune disease seen in lpr and gld mice.

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Effect of sirolimus on the metabolism of apoB100- containing lipoproteins in renal transplant patients.

BACKGROUND: Sirolimus (Rapamune, rapamycin, RAPA) is a potent immunosuppressive drug that has reduced the rate of acute rejection episodes by more than 40% in phase III trials when added to an immunosuppression regimen of cyclosporine (CsA) and prednisone. However, RAPA treatment tends to increase lipid levels, particularly among patients with pre-existing hyperlipidemia. METHODS: To identify the metabolic pathway(s) leading to RAPA-mediated hyperlipidemia, five patients with renal transplants maintained on CsA+/-prednisone+/- azathioprine (AZA) were studied before and after 6 weeks of treatment with RAPA (off RAPA and on RAPA, respectively). Each study patient was infused with a single bolus of [2H4]-lysine to derive metabolic parameters for apoB100-containing lipoproteins by using kinetic analysis based upon quantitation of isotopic enrichment by gas chromatography-mass spectrometry. RESULTS: Serial lipid measurements revealed that four patients displayed increased plasma triglyceride levels after RAPA treatment, which coincided with significantly higher plasma VLDL-apoB100 concentrations (21.7+/-12.1 mg/dl off RAPA vs. 38.7+/-14.8 mg/dl on RAPA, mean+/-SD, P<0.05). Kinetic analysis showed that the RAPA-induced increase in VLDL-apoB100 concentrations was due to a significant reduction in the fractional catabolic rate (FCR) of very low-density lipoprotein (VLDL) apoB100 (0.83+/-0.65 off RAPA vs. 0.24+/-0.10 on RAPA, mean+/-SD, P<0.05), rather than an enhanced VLDL-apoB100 synthesis. In one patient, RAPA treatment induced hypercholesterolemia but not hypertriglyceridemia. This hypercholesterolemia was due to elevated low-density lipoprotein (LDL) cholesterol levels, which coincided with a decreased FCR of LDL-apoB100. Heparin-induced lipoprotein lipase activity was significantly lower in the immunosuppressed hyperlipidemic patients than in normolipidemic controls. However, RAPA treatment did not significantly alter basal lipoprotein lipase activity in renal transplant patients in this study. CONCLUSIONS: This study indicates that for renal transplant patients in whom RAPA treatment induces hyperlipidemia, this effect is the result of reduced catabolism of apoB100-containing lipoproteins.

Carcinoma of prostate in men aged fifty and under: therapeutic options.

We made a retrospective study of 20 men, aged fifty or under, with adenocarcinoma of the prostate to evaluate presenting symptoms, stage, grade, and therapeutic results. Sixty-five percent were found to have extracapsular spread of disease (Stage C or D). The therapy used was one or a combination of three types: radical prostatectomy, radiation therapy, and hormonal manipulation. Five of 6 patients with Stage B disease and 3 of 6 patients with Stage C disease were treated with radiation therapy. The other Stages B and C patients underwent radical prostatectomy. In all 5 of Stage B patients receiving radiation, therapy failed; the mean time to tumor recurrence was 3.2 years. Two of 3 patients with Stage C disease died of metastatic disease within three years of receiving radiation. The 4 patients (Stages B and C) who underwent radical prostatectomy are free of disease. There was a statistically higher failure rate among the radiation therapy patients with Stages B and C disease than among the surgical patients (X2 = 8.4, p less than 0.1).

Use of a prostate model to assist in training for digital rectal examination.

OBJECTIVES: To assess the accuracy of prostate size estimation on digital rectal examination (DRE) before and after training with a three-dimensional prostate model relative to prostate volume by transrectal ultrasound (TRUS). METHODS: A total of 100 subjects underwent DRE by one of four family physicians (FP1, n = 34; FP2, n = 26; FP3, n = 22; and FP4, n = 18). One half were examined before any training on DRE prostate size examination and one half after the physicians were trained. Training involved teaching with a three-dimensional prostate model having posterior surface areas corresponding to the average dimensions of six different prostate volumes. The FPs were instructed to estimate the prostate size on the DRE to the nearest 5 g. A single urologist unaware of the DRE results performed TRUS on all patients to measure the prostate volume. RESULTS: Before training, the DRE size estimates ranged from 10 to 100 g (mean +/- SD 32.8 +/- 21.6), with a TRUS volume of 11 to 122 g (mean +/- SD 38.9 +/- 23.1). The correlation between the DRE and TRUS estimates was 0.25, suggesting low agreement (intraclass correlation coefficient [ICC] 0.35, 95% confidence interval 0.31, 0. 38). After training, 50 different patients had DRE size estimates of 10 to 100 g (mean +/- SD 39.4 +/- 19.7) and TRUS volume of 10 to 119 g (mean +/- SD 41.5 +/- 24.1). The correlation between the techniques was higher in patients examined after training (r = 0. 765), suggesting much better agreement between the techniques (ICC 0. 87; 95% confidence interval 0.86, 0.88). Among the physicians, agreement between DRE and TRUS was higher after training (ICC 0.64 to 0.96) than before training (ICC 0.02 to 0.49). CONCLUSIONS: Although the subjects examined before and after training differed, the agreement between TRUS and DRE prostate size estimates by the FPs appeared to be stronger after training with a three-dimensional prostate model. This model may be a useful tool to assist in training FPs and medical students to measure prostate size on DRE.

Limited angle reconstruction using stabilized algorithms.

The stability of solutions to the limited-angle tomography reconstruction problem obtained by using the projections-onto-convex-sets (POCS) technique are examined. Although POCS techniques provide a feasible solution to the reconstruction problem, the solution is only one sample from the intersection of the closed convex sets that define the solution space. A method for evaluating the ensemble of possible solution waveforms that are in the neighborhood of a solution is presented. The ensemble characteristics are used to construct an inverse filter which is then applied to the computed solution. The results obtained using this method are less sensitive to noise amplification and are less dependent on both starting data and the number of iterations. An estimate of the object-dependent extrapolation that is possible using either linear or nonlinear constraints is provided.

An explanation of the effects of Squashing in limited angle tomography.

The effects of Squashing, an algorithm introduced by J.A. Reeds and L.A. Shepp (see ibid., vol.MI-6, no.2, p.89-97, June 1987), are described. It is proven that Squashing induces a weight function upon the projections. Intuitive and numerical evidence is presented showing that this weight function is not desirable at limited angles. A method to counteract the effects of the weight function and extend the usefulness of the algorithm to 120 degrees is proposed.

Difference Fourier analysis of "surface features" of bacteriorhodopsin using glucose-embedded and frozen-hydrated purple membrane.

A difference Fourier map of the projected structure of bacteriorhodopsin has been synthesized from electron diffraction amplitudes collected from membranes prepared in the glucose-embedded state and the frozen-hydrated state. Phases of a recently published data set for glucose-embedded specimens were used for the difference Fourier map. Moderate resolution (9 A) and high resolution (4.25 A) maps both indicate that glucose is exchangeable for water in the region of the map corresponding to the lipid regions. We interpret this as indicating that there is a small surface depression in this region of the structure. The depth of this feature is estimated to be 1/6 the thickness of the protein region in the membrane. The data obtained in this study rules out the existence of an aqueous transmembrane channel, the dimensions of which are large enough to allow free exchange of glucose for water. Several new features are also observed in the protein region of the membrane. These features are probably due to segments of the polypeptide at the aqueous interface that are well ordered in frozen-hydrated specimens but not in glucose-embedded specimens. Candidate structures for the origin of these features are extensions of the helices, or linker regions connecting the helices.

Preparation of frozen-hydrated specimens for high resolution electron microscopy.

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.

Prevalence of low bone density in women with developmental disabilities.

This study was designed to assess the prevalence and severity of low bone mineral density in an institutionalized population of postmenopausal women (n = 79) with developmental disabilities, using quantitative ultrasound of the calcaneus. The results demonstrate that this population has a significantly lower quantitative ultrasound index (QUI) of the calcaneus compared with a referent population of age-matched control subjects without mental disabilities. We used the QUI T-score threshold of <-2.0 standard deviations (SDs) to define individuals at high risk for osteoporosis, and 82% of the study participants met this criterion. Furthermore, approx 43% of the population had a heel QUI that was more than 2 SDs below that of age-matched control subjects, consistent with a severe degree of demineralization.

Ureteral segment replacement using a circumferential small-intestinal submucosa xenogenic graft.

We wished to determine whether small-intestinal submucosa (SIS) will epithelialize when used as a ureteral replacement material. An 11-mm segment of native ureter was excised from eight New Zealand White rabbits and replaced with an 11-mm porcine SIS graft, which was circumferentially wrapped around a ureteral stent. The SIS ureteral grafts were harvested at 11 days or 35 days postimplantation and examined grossly and by standard light microscopy techniques. Partial epithelialization with the ingrowth of urothelium, smooth muscle cells, and blood vessels was observed in the grafts harvested at 11 days postimplantation. The SIS ureteral grafts examined at 35 days postimplantation showed additional restructuring of the smooth muscle cell layer and more organized epithelialization in comparison to the SIS graft examined at 11 days. After 35 days of regenerative healing, elements of all three layers of the native ureter were observed within the collagen matrix of the SIS graft. No significant complications were observed, but all subjects (8/8) demonstrated mild intra-abdominal adhesions. Mild collecting system dilatations were observed in 4/4 (100%) of the animals harvested at 35 days and in 0/4 (0%) of the animals harvested at 11 days. We have this demonstrated in this preliminary study that SIS xenografts will epithelialize when used as a ureteral replacement material. The repair mechanism of these ureteral grafts occurred through a regenerative healing process rather than by scar formation. With further studies, this material may prove to be a useful treatment option in patients with ureteral injuries.

An alternative to synthetic aortic grafts using jejunum.

Synthetic materials currently used for arterial grafts can provoke serious complications such as infection, rejection, and incomplete reendothelialization. Porcine small intestinal submucosa (SIS) allografts (homografts) may not share these disadvantages yet still may provide an effective graft alternative. This study was designed to provide additional information concerning the performance of this material when used as an allo-patch graft. A porcine model was selected to enable hemodynamic comparisons to the adult human physiology. Slaughterhouse small intestine was used to prepare the SIS graft material. A well-vascularized section ofjejunum was resected, rinsed, and maintained in 0.9% NaCl solution containing 10% gentamicin. The graft was prepared by removing the serosal layer, inverting the material, and discarding the mucosal surface in a similar manner. The remaining submucosa and stratum compactum constituted the graft material and was subdivided into sizes from 1.5 to 3.0 cm2 tacked to foil for manageability, and was returned to the gentamicin solution. The surgery was initiated with a femoral arterial line in the anesthetized swine, followed by isolation and preparation of the infrarenal aorta for grafting. Cross-clamps were applied and a 1.0 x 1.5-cm section of aortic wall was resected. Two sides of allograft were freed from the foil and sewn into the aorta. Then the remaining sides were removed from the foil and final graft placement was completed. The graft was harvested at postoperative day 28 and presented for microscopic examination. Results confirmed that reendothelialization with neovascularization had occurred during this interval.

Adaptive ultrasonic measurement of blood vessel diameter and wall thickness: theory and experimental results.

An adaptive ultrasonic technique for measuring blood vessel diameter and wall thickness is presented. This technique allows one to use a target-specific transmitted waveform/receiver filter to obtain a larger signal-to-noise ratio (SNR) in the received signal than conventional techniques. Generally, SNR of a received wave increases as the intensity of the transmit wave increases; however, because of the FDA limitations placed on the amount of transmit energy, it is important to be able to make the most efficient use of the energy that is available to obtain the best possible SNR in the received signal. Adaptive ultrasonic measurement makes the most efficient use of the energy that is available by placing the maximum amount of energy in the largest target scattering mode. This results in more energy backscatter from a given target, which leads to a higher SNR in the received waveform. Computer simulations of adaptive ultrasonic measurement of blood vessel diameter show that for a SNR of 0 dB in the transmitted waveform, the standard deviation of the diameter measurements for a custom-designed transmitted waveform is about two orders of magnitude less than the standard deviation of the diameter measurements using more conventional waveforms. Diameter and wall thickness measurement experiments were performed on a latex tube and a bovine blood vessel using both custom-made and conventionally used transmitted waveforms. Results show that the adaptively designed waveform gives a smaller uncertainty in the measurements. The adaptive ultrasonic blood vessel diameter and wall thickness measuring technique has potential applications in examining vessels which are either too deep inside the body or too small for conventional techniques to be used, because of the low SNR in the received signal.

Endometrial adenocarcinoma presenting in a premenopausal patient with tuberous sclerosis.

BACKGROUND: Endometrial adenocarcinoma is very uncommon in women under 40 years of age. CASE: A 39-year-old woman with tuberous sclerosis and severe intellectual disability presented with irregular bleeding unresponsive to oral contraceptive therapy. She was subsequently found to have a deeply invasive endometrial adenocarcinoma. CONCLUSION: Caregivers must pay particular attention to signs and symptoms in non-verbal patients. Persistent irregular bleeding on oral contraceptive therapy warrants additional evaluation.

Risk factors for low bone mineral density in individuals residing in a facility for the people with intellectual disability.

BACKGROUND: Individuals with intellectual disability (ID) are known to have a high prevalence of both low bone mineral density (BMD) and fractures with significant attendant morbidity. Effective strategies aimed at reducing fractures will be facilitated by the identification of predisposing risk factors. METHODS: Bone mineral density was measured by quantitative ultrasound of the calcaneus performed on 79 women and 132 men residing in a facility for adults with ID. Multiple variable logistic regression analysis was performed to determine the significance of risk factors for low BMD. RESULTS: Mobility impairment consistently appeared to be a significant risk factor for low BMD regardless of age or sex and especially for middle-aged men with profound ID. Further risk was identified for postmenopausal women taking enzyme inducing anticonvulsant medications and middle-aged men who were either smokers or tended to be short. Hispanic followed by Caucasian origin also put middle-aged males at a greater risk than their African-American counterparts. CONCLUSIONS: Specific risk factors for low BMD, some of which have potential for modification, were identified in the study population. Targeted strategies for risk factor reduction may result in a decrease in the high rate of fractures among these individuals.

Monte Carlo modeling of underwater-image formation: validity of the linear and small-angle approximations.

A Monte Carlo model has been used to compute a set of point-spread functions (PSF's) and modulation transfer functions (MTF's) that determine underwater-image quality in a range of different environments. The results have been used to analyze the range of application under which a linear-approximation theory holds. Conclusions of the study are that the linear-approximation theory Seems to hold quite well over a broad range of applications. The ramifications of the Wells small-angle-scattering theory that predicts the PSF from a knowledge of the volume-scattering function (VSF) are also considered.

Discrepancies are noted between a predicted and a computationally obtained MTF; these discrepancies increase with range. Therefore, the results of the simulations indicate that the small-angle-scattering theory is more valid at a limited number of attenuation lengths. The results of the simulations indicate that the theory is valid to approximately three attenuation lengths.

Cytokines and their role in the pathogenesis of severe food hypersensitivity reactions.

Cytokines are proteins produced by one cell that are capable of altering the behavior of another cell. Their biologic effects are both pleomorphic and redundant. A number of cytokines have been implicated in the genesis of allergic reactions, including IL-4, IL-5, IL-6, and tumor necrosis factor (TNF). In contrast, other cytokines such as gamma interferon (gamma-IFN) may have biologic actions that act to limit allergic responses. In studies on patients with severe food allergy, we have found cytokine production from peripheral T cells to be abnormal following mitogenic stimulation. These lymphokine abnormalities may contribute to the elevation in IgE and the eosinophilia seen in severe food allergy.

Three-dimensional ocean chlorophyll distributions from underwater serial-sectioned fluorescence images.

An underwater optical serial-sectioning technique is developed to measure in situ three-dimensional distributions of biological particles. The technique involves scanning of a thin plane of laser light through a range of distances parallel to the imaging plane of a digital CCD camera. Images of induced fluorescence in the sequentially illuminated planes are recorded. An inverse method is then used to reconstruct three-dimensional chlorophyll a distributions from the plane images. Computer simulations of the image formation and reconstruction process indicate that the underwater optical serialsectioning technique is practical for in situ determination and analysis of chlorophyll a microstructures for concentrations as low as 0.1 mg of chlorophyll a per cubic meter in 1--m(3) water volumes.

Low frequency sounds from sustained contraction of human skeletal muscle.

Low frequency audible vibrations are produced by human skeletal muscles undergoing sustained contraction. The effect is easily demonstrable with an electronic stethoscope which amplifies sound below 50 Hz. Autocorrelation analysis of the signal shows that it is periodic with a frequency 25 +/- 2.5 Hz. The quality of the sound is the same for all the skeletal muscles tested and is unaffected by changes in tension, ambient temperature, and blood flow. Electrically-stimulated contraction produces a sound which is indistinguishable from voluntary contraction. The amplitude of the sound increases linearly with tension. The sound signals are uncorrelated both in frequency and phase with electromyographic signals obtained simultaneously while the muscle is contacted. Arguments are presented to show that the sounds may be an intrinsic property of muscle contraction.