Radiation from tritiated thymidine perturbs the cell cycle progression of stimulated lymphocytes.

Tritiated thymidine was found to affect the cell cycle progression of phytohemagglutinin-stimulated human lymphocytes. By means of flow cytometry a statistically significant increase in the G2 and M phases of the cell cycle was observed in cultures with low concentrations of tritiated thymidine added 18 hours before the cultures were harvested.

Intra-operative parathyroid hormone monitoring in patients with parathyroid cancer.

BACKGROUND: Intra-operative parathyroid hormone (PTH) monitoring (IPM) is 97% accurate in predicting postoperative eucalcemia in sporadic primary hyperparathyroidism (SPHPT). However, its usefulness in parathyroid cancer has not been demonstrated. This study reports IPM accuracy during surgical resections for parathyroid cancer. METHODS: Eight of 556 consecutive patients with SPHPT underwent parathyroidectomy using IPM and had parathyroid cancer. Operative success was defined as eucalcemia > six months and operative failure/persistent cancer as hypercalcemia within six months of parathyroidectomy. The IPM criterion for operative success was defined as a >50% decrease of peripheral PTH levels from the highest either pre-incision or pre-excision values, 10 minutes after resection. RESULTS: In eight patients, 11 operations were performed. Ten operations (91%) resulted in >50% intra-operative PTH decrease. However, in only seven (70%) of these resections, eucalcemia was achieved for >6 months with five of these seven (71%) procedures being initial en bloc resections. The remaining 3/10 (30%) operations with >50% intra-operative PTH decrease resulted in operative failures. In the last operation, intraoperative parathormone monitoring (IPM) correctly predicted operative failure. IPM sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy in predicting outcome were 100, 40, 70, 100, and 75%, respectively. CONCLUSIONS: IPM with the criterion of >50% PTH drop from the highest level is less accurate in predicting operative success in parathyroid cancer when compared to SPHPT. A >50% intra-operative PTH level decrease in patients with parathyroid cancer, particularly in reoperative cases, is less predictive of complete resection. The initial recognition of this disease followed by proper resection remains essential in the treatment of parathyroid cancer.

Immunoprophylaxis and cytotoxic effector cells against EL 4 leukemia induced in syngeneic C57BL/6J mice by use of irradiated EL 4 cells.

Tumor-specific immunoprophylaxis was achieved in C57BL/6J mice against EL 4 leukosis cell challenge by sensitization of the syngeneic host with multiple ip injections of irradiated EL 4 cells. A minimal radiation dose was used to replication-block EL 4 cells before inoculation, as defined by dose-response analysis of irradiated EL 4 cells. Multiple ip injections of irradiated EL 4 cells stimulated development of significant, yet relatively low, levels of cytotoxic lymphoid activity (CLA) in lymphoid cells of the peritoneal exudate as measured by in vitro 51Cr-release cytotoxicity assays. The specific temporal and frequency dependencies of the inoculation regimen for achieving immunoprophylaxis indicated that, in addition to CLA, other, short-lived, immune processes were important in the tumor rejection. These observations showed the capacity of the C57BL/6J host for tumor-specific immune recognition and rejection of the syngeneic EL 4 leukemia. The tumor rejection could be elicited solely by inoculations of irradiated EL 4 cells and did not require exogenous amplifiers, such as immunoadjuvants, chemical modifiers, and/or allogeneic immune information transfer.

Flow cytometric analysis of R3327 rat prostate adenocarcinoma grown in vivo and in vitro.

The Dunning R3327 transplantable prostate adenocarcinoma in the Copenhagen rat is an acceptable model for the human disease. The G-subline (a rapidly growing carcinoma) and the H-subline (a slow-growing, well-differentiated adenocarcinoma) represent the extremes of differentiation and growth rate of this tumor. Both sublines were found to have one population that was diploid and a second aneuploid population that was hyperdiploid in DNA content. The percentage of hyperdiploid cells was significantly higher in R3327-G tumors than in R3327-H tumors. The tumor cell population ratios were stable in vivo, but the in vitro culture conditions supported only cells with diploid DNA content following four to five subcultures. These predominantly diploid cultured cells, when injected into intact male rats, resulted in tumors that had both diploid and aneuploid cells.

In vivo selection of androgen-insensitive cells in R3327-G rat prostate tumors: diethylstilbestrol diphosphate treatment versus orchiectomy.

Tumors grown in diethylstilbestrol diphosphate (DES)-treated rats grew significantly more slowly than tumors grown in orchiectomized animals, and tumors grown in orchiectomized animals grew significantly more slowly than tumors grown in controls (intact male rats). When these tumors (phase I) were dispersed and reimplanted into DES-treated, orchiectomized, or control rats in all possible combinations (phase II), a partial selection of androgen-insensitive cells was observed in tumors grown in DES-treated animals. Tumors grown in DES-treated phase I animals responded significantly less to DES treatment or orchiectomy in phase II. In contrast, tumors from phase I orchiectomized animals showed the same responses to orchiectomy in phase II. Since the administration of exogenous testosterone propionate prevented the growth rate inhibitory effects of both DES treatment and orchiectomy, the added effect of DES seemed to be antiandrogenic.

Tumor progression in serial passages of the Dunning R3327-G rat prostatic adenocarcinoma: growth rate response to endocrine manipulation.

Serial passages of the poorly differentiated, androgen-sensitive R3327-G prostatic adenocarcinoma were used to study the progressive changes that occur in tumor growth rate and androgen sensitivity. Different in vivo transplant generations (21st to 28th) were compared. The tumor doubling and animal survival times resulting from the implantation of the 21st to 22nd generation (21-22G) tumor cells in intact male rats were significantly greater than those resulting from the implantation of 23-28G tumor cells. The most dramatic difference between early (21-23G) and late (26-28G) tumor generations, however, was in androgen sensitivity. The 26-28G tumors displayed androgen sensitivity only when implanted into animals castrated 2 to 7 days previously. Tumors grown in the pretreated castrates grew at a significantly slower rate than those in intact rats and the pretreated castrates had longer survival times than the intact rats. When 26-28G tumors were allowed to grow in intact rats to approximately 1 cu cm and then the rats were castrated, no significant difference in the growth rate between these tumors and tumors grown in intact rats was observed. In contrast, the androgen sensitivity of 21-23G tumors could be demonstrated, regardless of whether treatment was started before or after implantation. The fact that androgen sensitivity was still evident under certain conditions in late-generation R3327-G tumors demonstrates that the basic mechanism involving androgen response was still present, although functioning at a much reduced level.

Hormone sensitivity of the R3327-G rat prostate adenocarcinoma: growth rate, DNA content, and hormone receptors.

The growth of the R3327-G rat prostatic adenocarcinoma was significantly reduced when implanted in orchiectomized male rats (ORCH tumors). Tumors grown in intact animals (control tumors) had a doubling time of 7.4 days as compared to 9.2 days in ORCH tumors. A computer-based analysis of flow cytometric DNA histograms also detected significant differences between control and ORCH tumors. ORCH tumors were found to have 25% fewer cells with hyperdiploid DNA than control tumors (p less than 0.01). This androgen sensitivity in growth rate and the proportion of hyperdiploid cells were further reflected in the binding of [3H]methyltrienolone ([3H]-R1881) to cytoplasmic (cytosol) and nuclear tumor extracts. ORCH tumor cytosols had a [3H]R1881 binding capacity which was 70% lower than controls (6071 fmol/g tumor tissue). Nuclear [3H]R1881 binding in ORCH tumors was undetectable in seven of eight samples while in control tumors, binding was detectable in five of six preparations. Sucrose density gradient analysis showed that cytosolic [3H]R1881 receptors sedimented at 8.1 S in low salt and 4.6 to 3.3S in high salt. Nuclear [3H]R1881 receptors in high salt sedimented at 4.1 to 3.3S. Competition experiments using [3H]R1881 showed that [3H]-R1881 receptors were primarily androgenic, although some displacement by estradiol did occur. In contrast, [3H]estradiol binding was found to be highly specific. The binding capacity of [3H]estradiol in ORCH tumor cytosols was 30% higher than controls (962 fmol/g tumor issue), while binding to ORCH and control nuclear extracts was similar. These data suggest that the inhibition of androgen-sensitive R3327-G tumor cells was related to the concentration of androgen receptors and that this in turn was expressed as a reduction in the proportion of hyperdiploid cells.

Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes.

A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.

Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation.

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.

Multiparameter kinetic analysis of killer cell initiation by using immune RNA.

By treating nonsensitized C57BL/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity which appeared as early as 8 hr after activation as measured by release of chromium-51 from labeled EL-4 target cells. Flow cytometric analysis and 3H-thymidine uptake data shown peak S phase activity at 72 hr. Maximum cytotoxicity was observed at 48 hr. Cell cycle kinetic parameters were correlated with the appearance of cell mediated cytotoxicity.

Efficient parathyroidectomy guided by SPECT-MIBI and hormonal measurements.

UNLABELLED: Parathyroidectomy is a difficult and lengthy operation which is noncurative in 6% to 10% of cases. To improve the efficiency of this operation, a new dual diagnostic approach was prospectively applied. METHODS: Preoperative tomographic 99mTc-sestamibi (MIBI) scintography and intraoperative measurements of circulating parathyroid hormone (PTH) levels by a quick assay (QPTH) were used. Scintigraphy comprised immediate and delayed planar and SPECT of the neck and chest, following 20 mCi MIBI. The presence and location of persistent foci of abnormal activity found within the neck mediastinum on volume-rendered reprojection (RPJ) of the SPECT data were reported. The surgion, guided by the three-dimensional MIBI-SPECT/RPJ images, identified and excised the single or most prominent scintigraphic focus and applied the QPTH. If PTH levels fell from baseline by at least 50%, the operation was concluded. RESULTS: The operative time of primary parathyroidectomy was reduced from an average of 90 min (before the introduction of scintigraphy and intraoperative PTH measurements) to 57 min. All but two patients became normocalcemic. In 58 consecutive patients with hyperparathyroidism, MIBI-SPECT/RPJ correctly and precisely identified 51 of 53 (96%) primary parathyroid adenomas, 14 to 15 secondary hyperplasias and 2 of 3 hyperplastic glands in MEN (sensitivity 94%, specificity 92%). QPTH verified the excision of the primary parathyroid adenomas and predicted normocalcemia in 50 of 52 patients. In 6 patients with misleading scintigraphy, QPTH was especially useful and guided the surgeon to continue the operation until the abnormal parathyroid tissue was found and excised. CONCLUSION: MIBI-SPEC/RPJ and QPTH sequentially applied improved the efficiency of parathyroidectomy.

Late parathyroid function after successful parathyroidectomy guided by intraoperative hormone assay (QPTH) compared with the standard bilateral neck exploration.

BACKGROUND: Controversy continues between bilateral neck exploration and limited parathyroidectomy. One approach depends on gland size and histopathologic factors; the other approach limits excision to only hypersecreting glands. Both have excellent early operative success, but late recurrence rates with limited exploration are unknown. METHODS: Three hundred twenty consecutive patients with primary hyperparathyroidism were followed 6 to 313 months after successful parathyroidectomy. One hundred seventy-six patients had bilateral neck exploration with excision of enlarged glands (group I); 144 patients had glands excised based on hyper-secretion (group II). Calcium and intact parathyroid hormone (iPTH) levels were measured yearly. Parathyroid gland hypersecretion was determined by elevated iPTH levels. RESULTS: In group I, 1 gland was excised in 160 patients (91%); 19 of 176 patients (11%) had elevated iPTH levels. In group II, 139 patients (97%) had 1 gland excised; 19 of 144 patients (13%) had high iPTH levels. The number of patients with more than 1 gland excised in group I (9%) is 3 times higher than in group II (3%) (P <.05). There was no significant difference in the incidence of recurrent hyperfunctioning glands between the 2 different operative approaches (chi-squared test). CONCLUSIONS: Late parathyroid gland function was comparable with both approaches. Multiple gland excision based on size alone may lead to excision of normal functioning glands.

Parathyroid metabolism after operative treatment of hypercalcemic (primary) hyperparathyroidism.

Parathyroidectomy is usually followed by a decrease in serum calcium, a lessening of symptoms, and a normocalcemic state that continues for years. Evaluation of parathyroid gland function after parathyroidectomy over a protracted period showed a continued hypersecretory state in many normocalcemic patients and is reported here for the first time. Patients identified with parathyroid hyperplasia (more than one gland excised) and patients who later developed mild renal failure were excluded. Seventy-seven patients undergoing parathyroidectomy with only one enlarged gland removed and the other normal-sized glands viewed or examined by biopsy were followed up from 5 to 16 years. Two patients developed recurrent hypercalcemia at 4 and 9 years after surgery. Seventy-five patients are considered "cured" and have normal serum calcium values. However, 28 (37%) of these normocalcemic patients have persistent elevations of parathyroid hormone. This increased parathyroid gland function suggests a continuing stimulation of the remaining glands. The rarity of clinical recurrence may be related to effective adaptations that prevent overt hypercalcemia. Many parathyroid adenomas appear to represent nonneoplastic disease.

DNA patterns in parathyroid disease predict postoperative parathyroid hormone secretion.

Flow cytometric analysis of the nuclear deoxyribonucleic acid (DNA) content of parathyroid glands excised from patients with hypercalcemic hyperparathyroidism has identified three distinct DNA patterns. The most frequent pattern showed a high percentage of cells with tetraploid DNA, which indicated an increase in the G2 and M phase of the cell cycle. Thirty-four patients were found to have abnormal tetraploid DNA content. One patient had a normal diploid pattern, and seven were found to have an aneuploid DNA population in their excised parathyroid glands. This unexpected finding of aneuploid DNA appears to be an unique feature of these endocrine glands because they have no histologic or clinical characteristics of malignant change. All patients have remained normocalcemic and clinically well after excision of only grossly enlarged glands. Postoperative parathyroid hormone (PTH) levels were correlated in 17 patients with DNA analyses of biopsy specimens from 30 normal-sized glands which were left in situ. Seven patients with elevated PTH postoperatively had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had been taken. In 10 patients with normal PTH levels, six had normal diploid patterns, whereas four had high tetraploid DNA in their gland biopsy specimens. DNA content present in biopsy specimens of normal-sized, in situ glands was predictive (p less than 0.042) of parathyroid gland secretory activity. These findings suggest that the stimulus for parathyroid gland hyperfunction often affects more than a single enlarged gland and persists after clinical cure, as shown by a more rapid cell turnover in some remaining glands and continued hypersecretion of hormone.

Clinical usefulness of an intraoperative "quick parathyroid hormone" assay.

BACKGROUND: Intraoperative assays of parathyroid hormone (PTH) in the surgical management of hyperparathyroidism have been limited by an extended "turnaround" time, making it impractical for the operating surgeon. With our modification of a standard immunoradiometric assay for intact PTH, results are reported in 12 minutes. The operative usefulness and the ability of this "quick PTH" assay to predict postoperative serum calcium levels are reported here. METHODS: Quick PTH levels from whole blood samples taken 10 minutes after excision of hyperfunctioning parathyroid glands were compared with preoperative and preexcision samples in patients undergoing 63 parathyroidectomies. Patients were divided into two groups with assay incubation times of 10 and 6 minutes. The latter was clearly not sensitive enough and resulted in a 20% false-negative rate. However, with a 10-minute incubation time, a decrease of 54% or more in quick PTH levels resulted in postoperative normocalcemia in patients with primary hyperparathyroidism. RESULTS: With these criteria used to predict the postoperative return to normocalcemia in 29 patients with primary hyperparathyroidism, the quick PTH assay had a sensitivity of 96%, specificity of 100%, and overall accuracy of 97%. CONCLUSIONS: The quick PTH assay was especially helpful in predicting postoperative calcium levels when multiple excisions were necessary to remove all hyperfunctioning tissue or some normal parathyroid glands were not visualized.

Incidence of multiglandular disease in primary hyperparathyroidism determined by parathyroid hormone secretion.

BACKGROUND: Successful parathyroidectomy depends on recognition and excision of all hyperfunctioning parathyroid glands. Because histologic definition is limited, multiglandular disease (MGD) is usually determined grossly by means of estimation of gland size and the experience of the surgeon, resulting in frequency varying from 8% to 33%. Normalization of elevated intraoperative intact parathyroid hormone (iPTH) levels after excision of all hyperfunctioning glands is necessary for postoperative normocalcemia and indicates normal secretion of remaining parathyroids. Abnormal hormone secretion measured during operation has been used to define the extent of excision and the incidence of MGD. METHODS: One hundred ten consecutive parathyroidectomy patients with no previous neck surgery or history of multiple endocrine neoplasia had intraoperative iPTH assays performed before and after excision of any suspected abnormal parathyroid gland(s). A drop in iPTH level after gland excision predicted postoperative normal calcium levels. RESULTS: All patients except one had normalization of serum calcium levels (average follow-up, 15 months). One hundred five patients had only one hyperfunctioning gland removed, and all have remained normocalcemic. Five (5%) patients had more than one gland involved: four had two or more hyperfunctioning parathyroids and one patient, who had a large parathyroid cyst removed, remained hypercalcemic. CONCLUSIONS: By using a biochemical assay, instead of estimated size, to predict which parathyroid glands are hypersecreting, the incidence of MGD in primary hyperparathyroidism was found to be 5%.

Familial pheochromocytoma.

During a 15-year period, 24 patients were treated operatively for pheochromocytoma at this medical center. In this group a father and three daughters were recognized to have the familial variety of this disease. Malignant hypertension, which began at an early age, was the most prominent characteristic. Operative treatment of the three sisters occurred at the ages of 9, 15, and 17 years. All pheochromocytomas were bilateral and were confined to the adrenal glands except for hepatic extension in the father. In contrast to most recent reports of familial pheochromocytoma, there was no evidence of multiple endocrine adenomatosis in this family. Total adrenalectomy was performed in only the youngest patient and, although normotensive, she requires close observation and steroid replacement. Three patients underwent a controversial partial adrenalectomy after excision of bilateral tumors. The father has remained hypertensive for 8 years and has evidence of a residual tumor in the liver. The two daughters who have had subtotal adrenalectomies remain normotensive, require no medication, and have normal urine catecholamine values 7 and 3 years after operation. These results show that removal of bilateral pheochromocytomas without total adrenalectomy may allow normal adrenal function free of recurrence for several years in patients without multiple endocrine adenomatosis.

Intraoperative parathyroid hormone monitoring as an adjunct to parathyroidectomy.

BACKGROUND: Parathyroidectomy has a success rate of greater than 95% in the hands of experienced surgeons. To maintain this result in a more cost-effective way, intraoperative monitoring of intact parathyroid hormone (iPTH) has been used to decrease operative times. This technique signals when all hyperfunctioning tissue has been excised or when further dissection is necessary. METHODS: Eighty-nine consecutive patients with hyperparathyroidism had plasma samples measured for iPTH levels during parathyroidectomy. Nine patients had previous neck explorations. Perioperative iPTH measurements using immunochemiluminescent assays with a turnaround time of 10 minutes were done after excision of each suspected abnormal parathyroid gland. RESULTS: All patients except one returned to and maintained normal calcium levels during the follow-up period of 8 months (range, 1 to 25 months). Prediction of postoperative calcium levels by means of quick immunochemiluminescent assay has a sensitivity of 97%, specificity of 100%, and an overall accuracy of 97%. Specific influence on surgical judgment was noted in four patients with multiglandular disease, in seven with difficult localization problems, and in one patient in whom the hyperfunctioning parathyroid tissue was not recognized. Monitoring the plasma iPTH levels during parathyroidectomy directly aided the surgeon's operative approach in these 12 patients. CONCLUSIONS: Intraoperative iPTH assay is useful with predictive accuracy of 97%. It influenced or changed the operative approach in 13% of patients.

The evolution of parathyroidectomy failures.

BACKGROUND: Reported operative failure rates for primary hyperparathyroidism range from 5% to 10%. Failure has been due to multiglandular disease, ectopic parathyroid glands, errors in frozen section, and missed diagnoses. Recently, our operative approach has changed from bilateral cervical exploration to direction by preoperative localization and intraoperative quick parathyroid hormone assay. The purpose of this study is to examine the causes and rates of failure in this evolving approach to parathyroidectomy. METHODS: Among 447 consecutive cases of primary hyperparathyroidectomy, 20 operative failures were examined. Three different operative approaches were compared with respect to causes and rates of failure. RESULTS: From 1969 to 1989, with bilateral neck exploration, failure was due to missed diagnoses, ectopic glands, multiglandular disease, and unknown causes, with a failure rate of 5%. From 1990 to 1993, with bilateral neck exploration and quick parathyroid hormone assay, failure was due to ectopic mediastinal glands, misinterpretation of frozen section, and operative judgment, with a failure rate of 10%. From 1993 to 1998, with preoperative localization and quick parathyroid hormone assay, the two operative failures (1.5%) were due to operative judgment and misinterpretation of the quick parathyroid hormone assay. CONCLUSIONS: The new surgical approach combining preoperative localization studies and intraoperative parathyroid hormone monitoring has eliminated the most common causes of parathyroidectomy failure and has significantly decreased the operative failure rate.