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L M Lee

Publications and source records attributed to L M Lee.

At least 37 records · Page 2Linked to original sources

XBMP-1B (Xtld), a Xenopus homolog of dorso-ventral polarity gene in Drosophila, modifies tissue phenotypes of ventral explants.

Previously we have isolated a Xenopus cDNA homolog of bone morphogenetic protein-1 (XBMP-1A). In the present report we describe a new cDNA clone called XBMP-1B (or Xtld) from a Xenopus embryonic library. Sequence analysis indicates that these two clones share an indentical N-terminal sequence, including a region of metalloprotease domain, three copies of a repeat first found in complement proteins C1r/s and an epidermal growth factor (EGF)-like sequence. XBMP-1B protein has an additional copy of an EGF-like sequence followed by two copies of complement 1 r/s repeat in the C-terminus. The overall protein structure predicted from the XBMP-1B sequence reveals that it encodes a protein homologous to Drosophila tolloid. Three XBMP-1 transcripts (2.9, 5.2 and 6.6 kb) were detected by northern blot analysis. However, the 2.9 kb transcript hybridized specifically with XBMP-1A and the 5.2 and 6.6 kb transcripts hybridized with XBMP-1B. In Drosophila, a major function of tolloid is to augment the activity of the decapentaplegic gene product, a close relative of tumor growth factor (TGF)-beta superfamily members, BMP-2/4. Although XBMP-1 and XBMP-4 are detected in various adult tissues of Xenopus, the expression pattern of these two genes was not tightly correlated. In the embryo, the expression of XBMP-1 increased gradually from the morula to the swimming tadpole stages. Injection of XBMP-1B RNA into the ventral blastomeres at the 4-cell stage caused an elongation of the ventral marginal zone explants and converted globin-positive blood cells to mesenchymal and muscle tissues at later stages. It was shown that XBMP-1A was less active and a 1A mutant lacking the signal sequence was inactive. Further studies revealed that injection of XBMP-1B RNA into the ventral marginal zone induced up-regulation of dorsal marginal zone markers, such as goosecoid and chordin, at the gastrulation stage. These data indicate that XBMP-1 may have a role in determining dorso-ventral patterning in Xenopus, but in a different way from the dpp/tolloid system demonstrated in Drosophila.

Amino Acid Sequence↗

Advantages of backscatter electron imaging scanning electron microscopy for intracellular localization of cardiac analytes by gold conjugated antibody.

Myoglobin and myosin light chain 1 (MLC1) are intracellular human cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.

Alkaline Phosphatase↗

A new method of measuring heparin levels in whole blood by protamine titration using a heparin-responsive electrochemical sensor.

OBJECTIVE: To determine the ability of a new electrochemical sensor to determine heparin levels in whole blood and to correlate the heparin levels as determined by this heparin-responsive sensor (HRS) with heparin levels as determined by the Hepcon assay system (Medtronic Hemotec, Parker, CO). DESIGN: Methods comparison study. SETTING: A large academic medical center. INTERVENTIONS: The heparin levels of 162 samples from 24 patients undergoing cardiopulmonary bypass were determined by the HRS system and by the Hepcon system. In 21 samples, heparin levels as measured by anti-Xa activity were determined as well. MEASUREMENTS AND MAIN RESULTS: HRS-determined values correlated highly with Hepcon-determined values (r = 0.942) and with anti-Xa determined values (r = 0.905). Bias +/- precision comparing the HRS and Hepcon methods was 0.211 +/- 0.478 U/mL. CONCLUSIONS: HRS determined that whole blood heparin levels correlate well with Hepcon-determined levels. These limited results indicate that further development and testing of this new technology are warranted.

Anticoagulants↗

Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis.

Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.

Amino Acid Sequence↗

Activation of nuclear factor kappa B in human neuroblastoma cell lines.

The nuclear factor kappa B (NF-kappa B) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF-kappa B translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor alpha (TNF alpha), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells NF-kappa B was present in the cytosol and translocated into nuclei as a result of TNF alpha treatment. The TNF alpha-activated NF-kappa B was transcriptionally functional. NF-kappa B activation by TNF alpha was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated NF-kappa B, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated NF-kappa B, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF-kappa B activation. We found, moreover, that in SK-N-Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF-kappa B was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF-kappa B activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNF alpha proved that NF-kappa B activation was not sufficient for induction of neuroblastoma differentiation.

Animals↗

Cisplatin-based chemotherapy for the treatment of advanced transitional-cell carcinoma of the urinary tract--a preliminary report.

The CMV (cisplatin, methotrexate, and vinblastine) and M-VAC (methotrexate, vinblastine, doxorubicin, and cisplatin) regimens were used to treat 19 patients with advanced transitional-cell carcinoma (TCC) of the urothelial tract. In the CMV group, the partial response rate was 45.5% and the mean response duration was 6.3 months. No complete response was obtained in our series. The median duration of survival was 15.8 and 8.3 months in responders and nonresponders, respectively. The toxic symptoms included one case of sepsis and three cases of renal toxicity. However, nausea and vomiting were experienced by most patients and required the administration of antiemetics. In the M-VAC group, the median duration of survival for responders was longer than that of nonresponders (greater than 10.2 vs 7.2 months), although the number of patients was too small for this difference to reach statistical significance. The toxic symptoms included one case of sepsis, two cases of renal toxicity, and nausea and vomiting in most patients. Bone metastasis in three patients did not respond to chemotherapy (CMV), a finding that is compatible with the results reported by other investigators. In summary, chemotherapy with the CMV or M-VAC regimen was effective in improving the response rate of patients. However, the duration of response was short, toxicity was severe in some cases, and the efficacy against bone lesions was poor. These problems must be solved to improve the outcome of patients with TCC following chemotherapy with the CMV or M-VAC regimens.

Adult↗

Renal colic in pregnancy.

Renal calculi are an infrequent but significant management problem during pregnancy. We reviewed all cases of renal colic occurring during pregnancy between 1979 and 1990 at Grace Hospital, a tertiary care obstetrical hospital in Vancouver, British Columbia. Of the patients 80 had a discharge diagnosis of renal colic and pregnancy during this 11-year period. Calculi were confirmed in 57 patients. Of the patients 66% were multiparous and 99% of the calculi occurred during either the second or third trimester. The most common symptom was flank pain seen in 89% of the patients, while greater than 95% displayed either microscopic or gross hematuria. Methods of radiographic diagnosis included ultrasonography and limited stage excretory urography. A total of 84% of patients passed stones spontaneously. Indications for urological or obstetrical intervention included persistent pain, sepsis, progressive hydronephrosis, solitary kidney or high grade obstruction. There were 37 procedures done in 23 patients. The most common procedure was placement of a ureteral stent. The complication rate associated with intrapartum intervention and stent passage in the 23 patients was 16%. All patients with a ureteral stent subsequently had spontaneous vaginal delivery without complication. A scheme for managing renal calculi in pregnancy is presented.

Adolescent↗

Adrenocortical insufficiency: a medical emergency.

Adrenal insufficiency is a deceptive disorder. Insidious in onset, chronic in nature, it can suddenly progress into an acute life-threatening condition that may mimic disorders of vastly different etiologies. The result can be a lethal delay in diagnosis. Prompt diagnosis and replacement of glucocorticoids and fluids are essential for survival. Acute adrenal insufficiency is frequently an exacerbation of an underlying chronic disorder of the adrenal cortex or pituitary gland. Yet any patient who has been treated with suppressive doses of glucocorticoids (e.g., cortisol, prednisone), experienced overwhelming sepsis, has received anticoagulant therapy, or has endstage metastatic carcinoma may suddenly develop adrenal insufficiency along with its deadly sequela of hypovolemic shock, hyperkalemia, hyponatremia, and hypoglycemia. Successful management of this condition requires not only a heightened clinical awareness of adrenal insufficiency, but effective stress reduction interventions and a thorough patient and family teaching program to support lifelong control of the disease.

Acute Disease↗

Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.

The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.

Animals↗

NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases.

NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha (TNF alpha). In the present work, we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNF alpha were able to activate NF-kappa B. Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNF alpha effect was unchanged. TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-kappa B in Jurkat cells. Thus, TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca(2+)-regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC, by a mechanism that increases NF-kappa B/I kappa B dissociation without affecting the NF-kappa B translocation step.

Calcium↗

Strain analysis of hepatitis B virus on the basis of restriction endonuclease analysis of polymerase chain reaction products.

To assess the value of classifying hepatitis B virus (HBV) strains at the genomic level with products from the polymerase chain reaction (PCR), an HBV PCR DNA typing procedure was developed. The design of this method was based on the selective sensitivity of the PCR product to digestion with different restriction endonucleases and on the size of the fragment resulting from a specific nuclease digestion. On the basis of published nucleotide sequences of different HBV subtypes, a set of primers was selected within the preS-S region. One of the primers was 1679F containing 25 nucleotides (5'-GGGTGGAGCCCTCAGGCTCAGGGCA-3'), and the other was 2254R containing 24 nucleotides (5'-GAAGATGAGGCATAGCAGCAGGAT-3'). All of the reactive sera produced the same sized 575-bp identification band. The product was subjected to digestion by a selected panel of restriction endonucleases. By using prototype HBV of known subtype as a model, these restriction nuclease maps of the PCR product were subtype specific. Most adr subtype clinical samples produced predicted PCR DNA restriction nuclease fragmentation in accordance with the nucleotide sequence of the prototype virus. Occasionally, samples of either the adw or the ayw subtype gave discrepant results in which they appeared to be a different subtype or were resistant to the restriction nuclease digestion. This genetic alteration was consistent in the paired specimens from sexual transmission cases. These results show that the described procedures are applicable to HBV strain assessment.

Base Sequence↗

Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells.

It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is involved in T cell activation and leads to autoimmunity. Although Class II antigen expression was found in various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells has not been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for the numbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages. None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosis characteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases; they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases, there were greater numbers of T cells per millimeter squared (229 +/- 94 [mean +/- the standard error of the mean]) than in the two HLA-DR-negative cases (37 +/- 1 [mean +/- range]). Three lacrimal gland specimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three specimens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands and cultured overnight in serum-free media supplemented with several potential mediators of Class II antigen expression: interferon-gamma, carbachol, or isoproterenol. Freshly isolated cells did not express Class II antigens at detectable levels, but in most experiments, they began to express the antigen even in the absence of putative mediators.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

Comparison between histopathology and DNA histogram of testis.

From January to July 1989, the DNA histogram of testicular open biopsies was done for 11 patients of primary infertility and 7 patients of a control group. There were 2 failures in these 36 specimens. The flow cytometric analysis revealed characteristic patterns in the relative numbers of haploid (1C), diploid (2C), and tetraploid (4C) cells. In the presence of normal spermatogenesis, the haploid compartment contained the majority of cells, followed by the diploid, and then the tetraploid (1C greater than 2C greater than 4C). The other diagnostic criteria of flow cytometry were as follows: hypospermatogenesis (2C greater than 1C greater than 4C), the maturation arrest (2C greater than 4C greater than 1C), and Sertoli-cell-only syndrome (2C, near 100%). According to the aforementioned diagnostic criteria, the results of DNA histograms were compared with the histopathology diagnosed by junior or senior pathologists. These patterns of DNA histograms correlated with the diagnoses by senior pathologists in 28 of 34 specimens (82.6%), while there were only 20 of 36 pathologic diagnoses (55.6%) which matched between junior and senior pathologists. It is shown that abundant experience is needed for testis pathologists to diagnose accurately. The DNA histograms correlate well with pathological findings, with the advantages of quantification and fewer specimens needed. In conclusion, testicular tubular cell DNA histograms appear to be a reliable modality in the evaluation of spermatogenesis. They provide the quantitative information of sperm maturation and help in making appropriate decisions in the management of male infertility.

DNA↗

Endoscopic balloon dilation of the prostate: early experience.

Coaxial balloon dilation catheters recently have been introduced for transurethral dilation of the prostate. We applied endoscopically a specially designed high pressure, large diameter balloon catheter to patients with benign prostatic hyperplasia or primary bladder neck hyperplasia who presented with symptomatic, cystoscopic and urodynamic evidence of obstruction. We report our preliminary experience with 42 patients: 28 with benign prostatic hyperplasia and 14 with bladder neck hyperplasia. Six months after dilation 46% of the patients demonstrated at least a 25% improvement in subjective (symptom score) and objective (corrected peak flow rate) parameters, while 6 (21%) experienced excellent (greater than 50%) symptomatic improvement despite unchanged corrected peak flow rates and 3 (11%) showed significant (greater than 50%) improvement in corrected flow rate alone. At 1, 3, 6 and 12 months the symptom scores decreased by 70, 67, 61 and 59% (median) from pre-treatment levels. Men with benign prostatic hyperplasia fared better than those with primary bladder neck hyperplasia (74 versus 58% decrease at 6 months). Objective evaluation by corrected peak flow rates demonstrated 25, 24, 28 and 2% median improvement at 1, 3, 6 and 12 months, respectively. Patients with primary bladder neck hyperplasia had substantially greater increases than those with benign prostatic hyperplasia (47 versus 27% at 6 months). Immediately after dilation the serum prostate specific antigen level increased by at least 1.5 ng. per ml. in 16 of 22 patients, indicating significant tissue trauma. Complications included mild bleeding and pain for the initial 24 hours and transient urinary retention in 2 patients. Our experience demonstrates the safety of an endoscopic approach to balloon dilation of the prostate that allows the urologist to maintain a central role in the assessment of a new approach to the treatment of obstructed voiding dysfunction.

Aged↗

Prophylactic bladder neck incision in the treatment of small benign prostatic hyperplasia.

From May 1986 to January 1989, a total of 178 patients with small prostate (less than 20 grams) received transurethral resection for benign prostatic hyperplasia. Twenty-two patients (12.4%) developed bladder neck contracture with an average interval of 247 days. The prophylactic bladder neck incision was performed concomitantly with transurethral resection of the prostate on 72 patients. Although smaller adenoma (average weight: 11.4 grams) was resected in this group, the bladder neck contracture occurred in 4 patients (5.6%), compared to 18 contractures in 106 patients (16.9%) who underwent transurethral resection of the prostate alone for larger adenoma (14 grams), P value less than 0.05. The operation time, peri-operative morbidity and hospital stay were similar in these two groups. It appeared that prophylactic bladder neck incision could protect against the formation of bladder neck contracture in patients with small obstructing prostatic adenoma.

Chi-Square Distribution↗

Prostaglandin E1 versus phentolamine/papaverine for the treatment of erectile impotence: a double-blind comparison.

The use of intracavernous prostaglandin E1 was studied in 48 organically impotent men. Eight men with previous chemical priapism did not have chemically induced priapism at up to 4 times the minimum effective dose of prostaglandin E1. Of 15 men with arteriogenic impotence who had failed prior intracavernous phentolamine and papaverine therapy 10 had adequate erections with prostaglandin E1. A total of 25 men received intracavernous prostaglandin E1 and phentolamine plus papaverine in a double-blind fashion. Erections with prostaglandin E1 were equal or superior to those with phentolamine plus papaverine in each case.

Alprostadil↗

[Use of double-J in extracorporeal shock wave lithotripsy of large renal calculi].

Treatment of large renal calculi by extracorporeal shock wave lithotripsy alone has not been accepted yet as the treatment of choice because of the high incidence of complications such as stone street inducing obstructive uropathy. From Sep. 1987 to Nov. 1987, Thirty four patients had received double-J catheter in ureter before E.S.W.L. to prevent ureteral obstruction. The age distribution was from 29 to 66 years old (mean = 49.1 Y/O) and male to female ratio was 3:1. All the stone size was larger than 20mm in its longest axial length. Each patient was followed at least 3 months, and the results indicated that 8 patients (23.53%) had stone free, 11 patients (32.35%) had sandy stone in kidney, and 15 patients (44.12%) had stone street in ureter. The additional treatments for significant residual stone included 3 patients needing ureteroscopy, 8 patients needing more than one session of E.S.W.L. and 4 patients having spontaneous stone passage after removal of double-J. Although the necessity of external diversion and the incidence of colic pain after E.S.W.L. would reduce so as to improve patients' quality of life, double-J does not increase the stone free rate, and ureteral obstruction was still possible to induce the renal function impairment. We suggest that double-J be removed as soon as possible if the stone fragments have no change in position after 2-4 weeks of followup and adjuvant treatment be necessary.

Adult↗