Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium.

Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO3) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.

The use of radiopharmaceuticals as an effective toxicologic technique for studying nephrotoxicity of drugs: cyclosporine-A.

The concept of altered biologic behavior of administered radiopharmaceuticals is used routinely in clinical nuclear medicine to increase the sensitivity of diagnosis, monitor the efficacy of chemotherapeutic drugs and radiation treatment, and determine injury caused by a drug whose effect has exceeded its therapeutic value. In this study, cyclosporine-A (CsA) an immunosuppressant drug known to cause nephrotoxicity due to tubular impairment and Tc-99m MAG-3, a renal imaging radiopharmaceutical secreted by the tubules have been used in animal models to establish a method for investigating the nephrotoxicity of drugs. New Zealand rabbits and Wistar rats were used. The rabbits and rats were treated with 30 mg/kg of CsA for 4 and 28 consecutive days respectively. Plasma creatinine and urea were measured and renogram studies were performed in the rabbits prior to and on 1, 4, 8, 11 and 15 days after treatment with CsA. For the renogram, the rabbits were given an intravenous bolus injection of 44.4 MBq (1.5 mCi) of Tc-99m MAG-3. The Tmax, T1/2, TTHM and uptake slope of the Tc-99m MAG-3 were calculated. Each rat was injected intravenously with 185 MBq (5 mCi) of Tc-99m MAG-3, killed 3 min later, the kidneys removed and 20 mm frozen sections made. Autoradiograms were generated from the frozen sections. Creatinine and urea levels were also measured in the rats. There was no consistent difference in creatinine and urea levels between control and CsA treated rabbits and rats. However, for the rabbit, on day 1 or 4 after treatment, there was significant increase in the values of Tmax, T1/2, TTHM and uptake slope between the control and CsA treated animals, indicating intrarenal vasoconstriction and delayed transit of Tc-99m MAG-3 from the parenchyma to the collecting system. This delay is dramatically shown in the tissue autoradiograms of the rats. The results are consistent with reported nephrotoxicity of CsA using other techniques. The results of this study, therefore, indicate that the concept of altered biologic behavior of Tc-99m MAG-3 can be used effectively as a toxicologic method for studying nephrotoxicity of drugs as exemplified by CsA.

Assessment of 99Tcm-tetrofosmin lung uptake: a modified method to avoid the contribution from high chest wall activity.

Evaluation of lung uptake during routine 99Tcm-tetrofosmin myocardial SPET (single photon emission tomography) studies may be hindered by substantial chest muscle uptake, particularly post-exercise. This study investigated this proposal and analysed the various components of chest activity that may add to the real lung uptake. Exercise SPET studies were performed on normal subjects using 99Tcm-tetrofosmin and compared with the results of 99Tcm-sestamibi and 201Tl studies. The chest to heart count ratio (CHR) was calculated from the anterior SPET projection and further subclassified into lung, anterior chest and posterior chest to heart ratios (LHR, AHR, PHR) from the left anterior oblique (LAO) projection. On post-exercise images, the 99Tcm-tetrofosmin CHR was significantly higher than that of 201Tl and 99Tcm-sestamibi. Both the 99Tcm-tetrofosmin AHR and PHR were higher than those of 201Tl and 99Tcm-sestamibi. However, the LHR was similar for all three radiopharmaceuticals. In contrast, the rest 99Tcm-tetrofosmin images differed little from the 201Tl and 99Tcm-sestamibi ones. Thus, 99Tcm-tetrofosmin lung uptake post-exercise should be interpreted with caution, as chest muscle uptake is higher than normal. A more accurate evaluation of 99Tcm-tetrofosmin lung uptake is achieved from the LAO projection, where the contribution from chest muscle counts is much less than in the routinely used anterior projection.

201Tl single photon emission tomography in the evaluation of residual and recurrent astrocytoma.

Twenty-five patients with malignant astrocytoma, either postoperatively (15 cases) or with recurrent tumour versus gliosis (10 cases) were included in this study. 201Tl single photon emission computed tomography (SPECT) was performed with the calculation of early and delayed uptake values and retention index. A high mean value of early and delayed uptake correlated with a low retention index in patients with high-grade astrocytoma in both postoperative residual and recurrent groups, versus a lower mean value of early and delayed uptake with a high retention index in patients with low-grade tumours. All postoperative cases with high-grade astrocytoma had high 201Tl uptake > 1.5, whereas 66.6% of cases with low-grade astrocytoma had low 201Tl uptake < 1.5. There was a correlation between retention index of 201Tl and tumour grade with r = 0.47. Also, recurrent cases showed remarkable differences in early and delayed 201Tl uptake (P < 0.05) and retention index (P < 0.001) compared with postradiation gliosis. There was a higher sensitivity in detection of tumour viability by 201Tl SPECT of 100% versus 80% using computed tomographic scanning and in the differentiation between recurrent tumour and postradiation gliosis.

201Tl scintigraphy in the staging of lung cancer, breast cancer and lymphoma.

To evaluate 201Tl in the detection of the primary tumour, lymph node involvement and mediastinal spread we have studied a total of 188 patients with histologically proven lung cancer, breast cancer or malignant lymphoma. Ten patients with benign lung disease were also examined. Static images were performed 20 min after intravenous injection of 75 MBq of thallous (201Tl) chloride. The results were compared with those of standard staging procedures including CT scanning and mediastinal exploration. Thallium-201 imaging was highly sensitive in detecting the primary tumour (lung cancer 86%, breast carcinoma 100%, lymphoma 85%), but showed low sensitivity in detecting mediastinal spread or lymph node involvement. Thallium-201 uptake was also observed in active sarcoidosis (one case) and active TB (two cases). We conclude that 201Tl imaging is unlikely to have a clinically useful role in the diagnosis or staging of lung cancer, breast cancer or lymphoma.

The optimum time for tumour imaging with thallium-201.

Following the intravenous injection of 75 MBq 201Tl-chloride we have assessed the uptake kinetics in the myocardium and in the primary tumour in 56 patients with lung cancer, 26 with breast cancer and 13 with mediastinal lymphoma. The time of maximal tumour uptake ranged from 8-20 min post-injection and did not differ significantly between lung cancer (mean +/- SD = 11.9 +/- 3.34 min), breast cancer (11.21 +/- 1.88 min) and lymphoma (11.76 +/- 3.25 min). The time of maximum cardiac uptake of 201Tl was 11.61 +/- 3.25 min. There was no significant washout of 201Tl from the tumours in the first hour after injection in the various malignant lesions studied. The time of maximal tumour to background activity was 18.3 +/- 0.59 min for lung cancer, 13.0 +/- 1.16 min for breast cancer and 16.7 +/- 1.04 min for lymphoma. The time course of 201Tl uptake in the tumours suggests that the mechanism of uptake is similar to that in the myocardium. The optimal time of 201Tl tumour imaging is from 20-60 min following injection and did not differ in various tumours studied.

Phase abnormalities in right heart studies. Demonstration of six different patterns.

Phase imaging abnormalities of the right side of the heart detected on multiple gated blood pool angiography (MUGA) have received less attention than similar abnormalities of the left ventricle. It has been found that certain different patterns of phase abnormalities of both right ventricle and right atrium are useful in the detection of six pathological conditions: right bundle branch block, ischemic right coronary artery disease, pericardial effusion, tricuspid regurgitation, pulmonary hypertension, and atrial septal defect. The authors emphasize the importance of these abnormal phase patterns during interpretation of gated cardiac studies, as they are helpful in directing the physicians attention towards the proper diagnosis.

"Pulmonary tongue" a right ventricle phase abnormality in muga studies in patients with pulmonary hypertension.

Pulmonary hypertension (PH) produces strain followed by hypertrophy and later dilatation of the right ventricle (RV) and pulmonary artery. The signs and symptoms are nonspecific. There is a need for a noninvasive sensitive way to diagnose PH. The purpose of this study is to evaluate phase abnormalities in radionuclide MUGA studies of patients with referred diagnosis of PH. In a retrospective analysis of 44 patients who had a radionuclide multigated study (MUGA) and contrast ventriculography (CV), 19 had high mean pulmonary pressure (over 20 mmHg) and a high pulmonary vascular resistance index (over 2.0). In 15 patients, a delayed phase segment in the RV corresponding to the pulmonary infundibulum and pulmonary conus was noted "The Pulmonary Tongue" sign (PT), 12 had PH (True positive) and 3 did not (false positive) on CV. No PT was seen in the remaining 29 patients, only 7 of them had PH (False negative). The sensitivity, specificity and accuracy of the PT sign in detecting PH was 80%, 72% and 77% respectively. The number of patients was too small to calculate the correlation of the grade of PT with the severity of PH. We conclude that "The Pulmonary Tongue" sign on a MUGA study is clinically useful in detecting PH.

Quantitative analysis of cardiac function: comparison of electro-cardiogram dual gated single photon emission tomography, planar radionuclide ventriculogram and contrast ventriculography in the determination of LV volume and ejection fraction.

A dual gated tomography (DGT) program for end systolic and end diastolic acquisition and subsequent processing for calculation of LVEF, end diastolic and end systolic volumes (EDV, ESV) has been evaluated in 20 healthy volunteers (25 years-40 years) and 45 patients (25 years-60 years): 20 with ischaemic heart disease and 25 with valvular heart disease (VHD). All had biplane multigated blood pool (MUGA) studies in the 40 degrees LAO projection using in vivo 99mTc- RBCs, immediately followed by DGT. The results in the patients group were correlated with contrast ventriculography (CV). In the volunteer group, the normal values for LVEF, EDV and ESV measured with DGT were found to be 63% +/- 10%, 91 ml +/- 6 ml and 30 ml +/- 6 ml and r value for the LVEF = 0.91 compared with MUGA. In the IHD group, r values compared with CV were 0.915 and 0.97 for the EDV and ESV and 0.934 for the LVEF. Compared with the MUGA, the r value for LVEF was 0.883. In the VHD group, r values were 0.98 for both the EDV and ESV and 0.948 for the LVEF (P less than 0.002) compared with CV and 0.789 for the LVEF compared with the MUGA. We feel that DGT is an accurate and reproducible technique for LV function measurements.

Radionuclide findings in arteriovenous fistula between left pulmonary artery and vein.

Right-to-left extra cardiac shunts are usually congenital and are rarely due to trauma or complicating thoracic or vascular surgery. A case of a right-to-left cardiac shunt due to an arteriovenous fistula between the left pulmonary artery and the left pulmonary vein is reported. This may be the first report of this abnormality. The etiology is not clear. The case was investigated only by radionuclide procedures because the general condition of the patient was so poor that invasive procedures could not be undertaken.

Detection of right-coronary-artery disease using stress thallium scintigraphy: importance of considering the right ventricle.

Retrospective analyses of the stress thallium studies of 51 patients with a history of previous infarctions or definite stable angina were performed in order to determine the extent of the coronary-artery disease (CAD). The results were correlated with recent coronary-angiography findings in order to assess the effect of including right-ventricule (RV) visualization on the accuracy of the detection of right coronary-artery (RCA) disease, as well as the effect of graded stenosis or occlusion of the RCA and the presence of disease of the other coronary vessels on the visualization of the RV. Eight patients had normal coronary arteries. The other 43 patients had CAD, only 5 of whom had single-vessel RCA disease. With regard to the RV, 6 patients had nonvisualization, 30 had patchy visualization, and 15 had normal visualization in the stress thallium studies. Nonvisualization indicated 100% occlusion of RCA in 6 out of 6 patients. Patchy visualization indicated significant occlusion of the RCA in 25 out of 30 patients (83%), while normal visualization indicated a normal RCA in 13 out of 15 patients(86%). Normal visualization did not mean that the possibility of RCA disease could be excluded. When RV visualization data were added to LV images, the sensitivity for detecting RCA increased from 66% to 94% (P less than 0.05), the specificity decreased from 89% to 72% (P less than 0.1), and the predictive accuracy increased from 75% to 86% (P greater than 0.1).

Clinical experience with stress and delayed thallium-201 quantitative segmental washout curves (QSWC).

Stress thallium-201 myocardial studies in 29 patients with chest pain were performed. The findings of digital display (DD) and quantitative segmental washout curves (QSWC) were compared independently and jointly with coronary angiography. It was found that QSWC is more sensitive (71%) than DD (55%) but is less specific (58%) than DD (98%). Taking both approaches together the sensitivity rises to 94% and the specificity falls to to 61%. Using both DD and QSWC for interpretation of thallium studies improves the agreement ratio with coronary angiography by 25% more than when using DD only.

Radionuclide study of the blood ocular barrier.

The purpose of this study was to investigate the permeability and clinical significance of the blood-ocular barrier. A new technique using systemic, subtenon, and retrobulbar injections was applied to the study of intraocular penetration of labelled steroid compound. The study was carried out in normal rabbits and in animals with artificially induced intraventional inflammation. It is concluded that there is no blood-vitreous barrier, a vague concept in any case, in the anterior region of the vitreous where free diffusion was observed between the anterior and posterior segments of the eye. High concentration of the labelled steroids was found in the cornea with the peak of activity usually at 30 minutes. However, traces of activity were detectable for up to 4 hours. This indicates the need for a depot long term respiratory form of corticosteroid that would deliver a high concentration of the medication. Local subtenon's and retrobulbar injections resulted in a relatively higher ocular I-125 Cortisol concentration than obtained by systemic route. It is concluded therefore that a high concentration of steroids applied locally will give better and less deletrious effects than systemic administration. Subtenon injection resulted in higher activity in various ocular tissues especially aqueous and vitreous. However, the I-125 Cortisol was rapidly cleared regardless of the route of administration.

Scintigraphic detection of acute gastrointestinal bleeding using 99Tcm-DTPA.

Technetium-99m DTPA is a good agent for detecting sites of acute gastrointestinal bleeding due to its rapid clearance by the kidneys and its stability in the bowel achieving a good target to nontarget ratio. It has been successfully tried for a pilot study. Further evaluation is in progress for its sensitivity. In cases where there is a slow rate of bleeding, we are currently evaluating injection of the dose by infusion, slowly over 15 min instead of the rapid i.v. injection.

Radionuclide scanning of the spleen. Technical modification, normal variants and dimensions.

In order to cut down the time needed for the preparation of the thermally damaged erythrocytes, the effect of heat was combined with the use of a higher concentration of ACD. Heating of the 51Cr tagged red cells was performed in a water bath, at 50 degrees C for 15 minutes, in the presence of ACD in the ratio of one ml ACD for every 3 ml blood. Using this technique, the obtained splenic scans in 20 normal individuals were excellent in quality. In the posterior view, the spleen was oval with an oblique long axis in 65% of the cases and transverse long axis in another 15%. In the remaining 20% the spleen was globular. The average measurements of the spleen were 10.5 X 7,1 cm, with a circumference of 29.8 cm. By planimetry, the surface area of the spleen averaged (59 +/- 12.4) cm2 (Mean +/- 1 S.D.) In the lateral projection, the surface area of the splenic scan was larger by an average of 40% than the picture obtained through the posterior approach in all the six examined subjects.