Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

Application of DNA amplification to pneumocystosis: presence of serum Pneumocystis carinii DNA during human and experimentally induced Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). Much remains unknown about the basic biology of P. carinii and studies of this infection have been hampered by the lack of cultivation methods. We developed a sensitive and specific assay for P. carinii by utilizing DNA amplification of the P. carinii dihydrofolate reductase (DHFR) gene. By this method, P. carinii DNA was detected in the lungs of rats with experimentally induced P. carinii pneumonia 2 wk before the onset of histopathological changes. DNA amplification analysis of serum demonstrated that by 10 wk of corticosteroid treatment, 12 of 12 (100%) infected rats had circulating DHFR DNA. P. carinii DHFR DNA also was detected in the serum of patients with AIDS and active P. carinii pneumonia (12 of 14 sera collected prospectively). Patients with advanced AIDS but without a history of P. carinii pneumonia were negative by this assay (0 of 6 sera examined). Serum polymerase chain reaction may facilitate investigations into the natural history and epidemiology of P. carinii infection, provide insight into the pathogenesis of parasite dissemination, and offer a useful, noninvasive diagnostic test for the detection of human pneumocystosis.

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii.

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.

Transport of ethanolamine and its incorporation into the variant surface glycoprotein of bloodstream forms of Trypanosoma brucei.

The membrane-attached form of the variant surface glycoprotein (mf-VSG) of bloodstream forms of Trypanosoma brucei is anchored to the plasma membrane by a hydrophobic C-terminal lipo-oligosaccharide containing ethanolamine. Analysis by polyacrylamide gel electrophoresis showed that several different cloned T. brucei strains (strain EATRO 110 and variants 117 and 118 of strain 427) incorporated [3H]ethanolamine into both mf-VSG and the soluble VSG derived from it, but not into other proteins. Other trypanosomatids, e.g. Leishmania mexicana promastigotes, T. cruzi epimastigotes, and T. brucei procyclic forms, did not incorporate ethanolamine into cellular proteins. Thus, [3H]ethanolamine can be used as a specific biosynthetic label for T. brucei VSG polypeptides. The time course of incorporation of [3H]ethanolamine into VSG showed a lag period of about 15 min. Double-labelling experiments using [3H]ethanolamine and H3[32P]O4 demonstrated that ethanolamine labelled only the C-terminal moiety and was not incorporated into other portions of the VSG molecule. Cellular uptake of ethanolamine occurred via a specific carrier-mediated transport system having a Vmax of 132 pmol min-1 mg-1 protein and a Km of 3.7 microM. The properties of this transport system are consistent with the possibility that ethanolamine is derived entirely from the host.

Human serum-sensitive Trypanosoma brucei rhodesiense: a comparison with serologically identical human serum-resistant clones.

Trypanosoma brucei rhodesiense clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis is indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.

High-density-lipoprotein-independent killing of Trypanosoma brucei by human serum.

The cattle pathogen Trypanosoma brucei brucei is morphologically indistinguishable from the human pathogens T.b. rhodesiense and T.b. gambiense. However, unlike the human pathogens, T.b. brucei is lysed by normal human serum (NHS). The trypanolytic factor in NHS co-purifies with high-density lipoproteins (HDL), but its precise nature is unknown. Using a new fluorescence-based viability assay to assess T.b. brucei killing, we find that the HDL-deficient sera from two patients with Tangier disease are as trypanolytic as NHS. Fractionation of the Tangier sera by density ultracentrifugation revealed that the activity resides only in lipoprotein-depleted fractions. Tangier and NHS were also subjected to molecular sieving chromatography, and the activity profiles were identical. Lytic fractions to T. brucei (but not to T. rhodesiense) appeared under two distinct peaks of 100-600 kDa and > 1000 kDa. Neither peak coincided with the position of the major serum lipoproteins, as determined by cholesterol titrations. The high-molecular-mass peak did not contain the HDL-associated apolipoprotein-A1. Further, we did not find that purified apolipoproteins A1 or J are lytic for the trypanosomes. We conclude that the killing of T. brucei by human serum can be independent of HDL.

Needle biopsy DNA ploidy status predicts grade shifting in prostate cancer.

DNA ploidy analysis of prostate needle biopsy specimens was performed to determine whether ploidy status could predict tumor grade shifting at radical prostatectomy. The paired needle biopsy and radical prostatectomy specimens from 111 randomly selected men with prostate cancer were obtained from the surgical pathology files of the Albany Medical Center Hospital. The original tumor grades were assigned by a staff of 12 surgical pathologists according to the Gleason system. Tumors with original Gleason scores < or = 6 were classified as low grade, and tumors with scores of > or = 7 were considered high grade. DNA ploidy analysis was performed on the needle biopsy specimens using the CAS 200 image analyzer (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) on Feulgen stained 5-microm tissue sections. There were 88 diploid and 23 nondiploid cases. Thirty-eight of 111 (34%) of cases had grade shifting from needle biopsy to radical prostatectomy specimens. Of 89 low-grade needle biopsy cases, 28 (31%) were upgraded at radical prostatectomy. Of 22 high-grade needle biopsy cases, 10 (45%) were downgraded to low grade at radical prostatectomy. Of the 28 low-grade needle biopsy specimens that were upgraded at radical prostatectomy, 19 (68%) featured an aneuploid histogram and 9 (32%) were diploid. Nineteen of 28 (68%) of aneuploid low-grade tumors on needle biopsy became high-grade at radical prostatectomy. Nine of 10 (90%) diploid high-grade tumors at needle biopsy became low-grade at radical prostatectomy. Of the 38 cases in which ploidy and grade were incongruous, 28 (74%) had grade shifting. In a multivariate regression analysis, a high-grade Gleason score on radical prostatectomy specimens correlated significantly with needle biopsy ploidy (p = 0.0001) but not with needle biopsy grade (p = 0.15). The sensitivity of the needle biopsy grade in the detection of high-grade tumors on radical prostatectomy was 30%, and the specificity was 86%. The sensitivity of ploidy status in the prediction of high grade at radical prostatectomy was 78%, and the specificity was 96%. With a prostate-specific antigen (PSA) level of >0.4 ng/ml as the indicator of post-radical prostatectomy disease recurrence on a subset of 106 patients, on univariate analysis, disease recurrence was predicted by needle biopsy ploidy (p = 0.001) and radical prostatectomy grade (p = 0.04) but not by needle biopsy grade (p = 0.39). On multivariate analysis, needle biopsy DNA ploidy status independently predicted disease recurrence (p = 0.002), whereas needle biopsy and prostatectomy grade did not. These results indicate that DNA ploidy analysis of needle biopsy specimens of prostate cancer predicts grade shifting, that it is a more sensitive and specific indicator of final tumor grade at radical prostatectomy than is the original needle biopsy grade, and that ploidy status independently predicts postoperative disease recurrence.

Magnetic resonance chemical shift imaging and spectroscopy of atherosclerotic plaque.

An in vivo magnetic resonance imaging (MRI) technique for identification and characterization of atherosclerotic plaque was assessed in animal and human models. Atherosclerosis was induced in the abdominal aorta of four rabbits by a combination of balloon denudation and a high cholesterol diet. In vivo conventional spin-echo and fat/water suppressed images of the rabbit aortae were obtained at 1.5 T. Chemical shift imaging (CSI) was achieved using a hybridization of selective excitation and modified Dixon techniques. These techniques were then used to obtain images of atherosclerotic lesions in the carotid arteries of four patients prior to endarterectomy. The MRI results were corroborated by histologic and high-resolution proton MR spectroscopic (8.5 T) analysis of rabbit aorta, human carotid endarterectomy, and six additional human superficial femoral and iliac atherectomy specimens. All animal and human lesions were classified as either fatty streaks or fibrotic plaque. When compared to conventional spin-echo images, fat suppression by CSI substantially improved the measured contrast-to-noise ratio between plaque and vessel lumen, and enhanced its discrimination from periadventitial fat. In contrast, water suppression eliminated visualization of plaque due to the negligible amount of isotropic (liquid-like) signal from the immobilized lesion lipids. Magnetic resonance spectroscopy corroborated the CSI results by demonstrating broad, ill-defined fat resonances characteristic of nonmobile lipids in both human and rabbit atherosclerotic lesions. These findings indicate that in vivo MRI of plaque is technically feasible and can be markedly improved using chemical shift imaging.

Characterization of benign and malignant tissues of the thyroid gland. An ultrasonic approach using RF waveform analysis and pattern recognition.

A physical basis was developed for an accurate noninvasive technique to differentiate malignant thyroid tumors from benign lesions based upon a frequency-dependent attenuation model. The research effort utilized RF waveform analysis via a minicomputer based digital processing system. Data collection was performed using a standard clinical B-Mode diagnostic instrument with a transducer specially designed for imaging the thyroid gland. Differences between the frequency spectra of the RF waveforms for benign and malignant pathologies could be demonstrated in accordance with the proposed model.

Fatty liver. Chemical shift phase-difference and suppression magnetic resonance imaging techniques in animals, phantoms, and humans.

In vitro animal and human models were used to evaluate the potential of chemical shift magnetic resonance imaging (MRI) for assessing fatty liver. Phantoms of varying fat content were created from mayonnaise-agar preparations. Fatty liver was induced in eight rats by feeding them ethanol for three to six weeks (36% of total calories), whereas eight control rats were fed a normal diet. T1-weighted in-phase and opposed-phase MR images were obtained of the phantoms animals, and 28 human subjects. Additional images obtained in animals included long TR images with in-phase and opposed-phase technique, and hybrid chemical shift water and fat suppression. The rats were killed and histologic status was graded blindly by a hepatopathologist as normal, mild, moderate, or severe fatty change, for correlation with MR grading. Quantitative analysis of MR images included fat signal fraction for animals, and relative signal decrease between in-phase and opposed-phase images for phantom and human data. Phantom in-phase signal increased linearly with respect to fat content, whereas opposed-phase signal decreased linearly. MRI and histologic grading of rat livers were highly correlated, especially when based on water suppression images (r = 0.91, P = .0001). Opposed-phase images were also highly correlated, while fat suppression images were less effective. There was no overlap between MR-derived fat fractions for control (2.6%-5.7%) versus ethanol-fed rats (7.7%-17.9%, P = .0002). Human liver considered to be fatty by visual inspection (n = 8) had higher relative signal decrease than nonfatty liver (n = 22) (P less than .001). Phantom, animal, and human data demonstrate that comparison of T1-weighted in-phase and opposed-phase images is both practical and sensitive in the detection and grading of fatty liver.

Quantitative immunohistochemical determination of cathepsin D levels in prostatic carcinoma biopsies. Correlation with tumor grade, stage, PSA level, and DNA ploidy status.

National screening programs resulting in an increased detection rate of prostatic adenocarcinoma have prompted the search for new methods of predicting disease outcome that can be applied to the initial narrow bore needle biopsy specimens. Cathepsin D, a lysosomal aspartyl protease and autocrine mitogen, has been studied in a wide variety of human neoplasms as an invasion and metastasis marker. Prostatic carcinoma needle biopsy tumor cell cathepsin D content was measured in 61 men using a semiquantitative image analysis assisted immunohistochemical procedure. Results were compared with preoperative serum prostatic specific antigen levels, tumor grade, DNA ploidy status, pathologic stage after radical prostatectomy and disease recurrence during a median 2.6 year follow-up. Biopsy cathepsin D levels significantly correlated with tumor grade (P = .022) and DNA ploidy status (P = .028) by logistic regression analysis. Post-prostatectomy pathologic stage and disease recurrence did not correlate with tumor cathepsin D levels. Final prostatectomy grade and DNA ploidy status independently predicted metastasis and post-operative disease recurrence (P < .001). Although this study did not find independent prognostic status for cathepsin D in prostate cancer, the correlation with tumor grade and DNA ploidy status is noteworthy and the inter-relationship of outcome variables may prove of interest and warrant further evaluation of this potential predictor or CO-predictor of disease outcome.

Ultrasonography: an alternative to x-ray-guided needle localization of nonpalpable breast masses.

Preoperative ultrasonography was used as an alternative to x-ray mammography to localize 92 breast lesions encountered in 82 patients. Recommendation for biopsy was made on the basis of the ultrasonographic finding of a nonpalpable mass or an area of architectural distortion, or in the presence of equivocal physical findings if sonomammography demonstrated a solid or an anechoic mass. Sonomammography was performed in the operating room, just before anticipated biopsy, with a hand-held high-resolution scanner. When the suspicious area was imaged and its precise location noted, the breast was then prepared and draped in the usual manner, and a biopsy was performed. If the suspicious area could not be easily localized after the incision was made and the breast explored, the transducer was "gowned" and used directly in the wound to help find the lesion. This technique has proven effective and accurate. In selected patients ultrasonography may be used as well as, or instead of, x-ray needle localization for the precise excision of nonpalpable breast lesions, excluding calcifications.

Ureteral ectopia presenting as epididymitis and infertility.

A thirty-two-year-old man presented with a history of infertility, recurrent epididymitis, and a fluctuant pelvic mass on rectal examination. Preoperative evaluation disclosed a nonvisualized right kidney and a multiloculated cystic lesion in the pelvis. The intravenous pyelogram suggested a ureterocele on the right side. A cystic pelvic mass was noted on ultrasound and computed tomography. A dysplastic right kidney with an ectopic ureter inserting into a cystic seminal vesicle was found at surgical exploration.

Ultrasonography of the lower genitourinary tract.

As techniques and equipment become even more sophisticated, the use of diagnostic ultra-sound should expand. More accurate diagnoses, simplification of invasive techniques, and increased benefit of therapeutic techniques can be expected.

Ultrasound examination of the hand and foot.

The use of ultrasound in the examination of the distal extremities is a recent application of sonography of the musculotendinous system. Real-time sonography is unique in providing a dynamic study of complex anatomic structures such as the flexor tendons of the hand. The normal anatomic appearance of the hand and foot are reviewed as well as the sonographic appearance of pathologic conditions of the distal extremities.

Ultrasound examination of tendons.

This article reviews the type of equipment used in sonography of the tendons, the method of approach in viewing normal tendon anatomy, and the sonographic appearance in pathologic conditions of the tendons. The role of ultrasound in follow-up studies is also emphasized because of its noninvasive nature.

Recent advances in magnetic resonance imaging of the knee.

The examination of the knee has rapidly become the most important non-neurologic application of MR imaging. The widespread availability of high signal-to-noise knee coils has made routine imaging with T2-weighted sequences in both coronal and sagittal planes possible in 30 minutes. The spin-echo sequence remains the most important imaging technique, although many newer sequences have also been applied to the knee, with varying degrees of success. Important pitfalls in diagnosis, such as high signal intensity in the posterior horn of the medial meniscus and the transverse meniscal ligament, displaced buckethandle meniscal tears, and discoid menisci, can be recognized with experience. Common clinical problems that are encountered include meniscal cysts, osteonecrosis, and bone bruises. Detection of subtle injuries of the medial collateral ligament, patellar tendon, and anterior cruciate ligament requires careful observation. Intra-articular loose bodies can be reliably detected with MR imaging, and it should be recognized that localized pigmented villonodular synovitis can sometimes simulate the appearance of a loose body. MR imaging is a valuable noninvasive procedure that is complementary to arthroscopy in the evaluation of diseases of the knee.

Magnetic resonance imaging of prostate cancer: update.

Prostate cancer is the most common cancer in the world, the most frequently diagnosed in the United States, and the second most lethal cancer in U.S. men. Earlier diagnosis implies better prognosis. However, prognosis may be dependent upon the stage of the malignancy at the time of diagnosis and implementation of appropriate therapy. Clinical staging, even with the development of serum prostate-specific antigen and other studies, has not proven to be highly accurate, particularly to identify and quantitate local disease and extension. Imaging has, in the past, also had limited success. With the development of computed tomography (CT), endorectal ultrasound, and magnetic resonance imaging (MRI), there was great expectations for improvement. However, CT and ultrasound have not been as accurate as hoped. MRI, because of its multiorientation and multiparameter abilities, has been the most definitive imaging tool for staging of local extension, yet still has limitations. The prostate capsule, the neurovascular bundles, the seminal vesicle, and other regions prone to initial attack by cancer extension can be seen exquisitely clearly by the newer approaches to MRI. Cancer extension, however, cannot be consistently identified when it is microscopic. MRI is an accurate identifier of macroscopic, even subtle macroscopic disease, but there are still limitations in its ability to diagnose all pathology.

Magnetic resonance imaging of the prostate and bladder.

Prostate carcinoma is the most common cancer in men and the second most lethal malignancy among the American male population. Nevertheless, it is potentially curable if detected early and treated appropriately. Treatment options vary depending on the extent (stage) of the cancer. MRI has no role as a screening method for prostatic carcinoma because it is expensive, time consuming, and unable to differentiate benign from malignant disease, but it can detect early prostatic cancers in patients with known tumor and can accurately stage these tumors. MRI is becoming the imaging modality of choice for local staging of prostate cancer and is rapidly replacing CT and ultrasonography for this purpose. Treatment protocols for bladder carcinoma also depend on the stage of the tumor. Clinical staging of bladder cancer has been limited. MRI is as good as or better than CT in bladder tumor staging when extravesical tumor involvement is present. MRI also has the potential to become useful in determining the depth of wall invasion in tumors confined to the bladder.

Multiple spin-echo MR imaging of the body: image contrast and motion-induced artifact.

For a given TR and TE, image quality changes when the number of spin echoes obtained is varied. To investigate the importance of this in clinical imaging, a total of 4 patients and 9 volunteers had MRI examinations of the abdomen (n = 7) and/or pelvis (n = 8) which included at least 2 sequences with identical TR (2000 or 2500 ms), TE (80 ms) and other parameters, but with a different series of refocusing pulses. Sequences included single-echo (S), asymmetric and symmetric double-echo (AD and SD) and quadruple-echo (Q) techniques. Image contrast and severity of motion-induced artifact was measured via blind examination by 3 independent MRI radiologists and calculation of signal-difference, signal-difference-to-noise ratios and intensity of motion-induced "ghost artifact." The order of decreasing signal differences was S, SD, AD and Q, and all of three liver lesions were better seen with S than with SD techniques. These observations are consistent with signal loss from cumulative inaccuracies from multiple 180 degrees RF pulses. The order of increasing intensity of ghost artifact was Q, SD, AD and S, consistent with the beneficial motion artifact-reducing effects of even-echo rephasing. Knowledge of these effects of multi-echo imaging allows one to make informed decisions about imaging protocols rather than to simply obtain multiple echoes "because they are free."