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Background: The purpose of this study is to report the minimum 5-year clinical and radiographic outcomes of patients undergoing anatomic total shoulder arthroplasty (aTSA) with a posteriorly augmented glenoid component., Methods: Thirty-five shoulders with minimum 5-year follow-up underwent aTSA using a posteriorly augmented glenoid component for the treatment of glenohumeral osteoarthritis with posterior glenoid bone loss. Clinical outcomes included range of motion, visual analog scale (VAS) for pain, and patient-reported outcomes scores. Radiographs and computed tomography scans were obtained to assess glenoid morphology, retroversion, and central peg osseous integration via Wirth score, Lazarus scores, and Yian scores. Postoperative outcomes were measured at minimum 2- and 5-year follow-up. Kaplan-Meier survival analysis was calculated., Results: Two patients experienced prosthetic instability requiring revision, leaving 33 shoulders with an average follow-up of 6.6 years. Average preoperative glenoid retroversion was 21.6°. A significant improvement in pain, range of motion, and patient-reported outcomes score was noted at minimum 2- and 5-year follow-up. In addition, VAS pain, American Shoulder and Elbow Surgeons, and Quick Disabilities of the Arm, Shoulder, and Hand scores improved between minimum 2- and 5-year follow-up. The average Lazarus and Yian scores at final follow-up were 0.73 and 2.6, respectively. There was an increase in Lazarus score and a decrease in Wirth score between 2- and 5-year follow-up. A significant correlation was identified between VAS pain scores and both Lazarus and Wirth scores. Survivorship free from revision was 92% at 8.8 years postoperatively., Conclusions: Midterm results of aTSA with a posteriorly augmented, stepped glenoid component demonstrate sustained improvements in clinical outcomes with low rates of radiographic loosening. Continued improvement in pain and function, as well as a minor progression of radiographic osteolysis, may be expected between 2- and 5-year follow-up. In addition, the severity of radiographic loosening correlates with subjective pain levels., (Copyright © 2024 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Medial clavicle fractures pose a concern for mediastinal compromise because of their proximity to the sternoclavicular joint. However, the true incidence of this complication is unknown. The purpose of this study was to evaluate fracture configuration and determine the incidence of mediastinal compromise following medial clavicle fractures. A retrospective analysis of all patients treated for isolated medial one-third clavicle fractures at a single institution was performed. Patient demographics, the mechanism of injury, complications, and treatment were recorded. The fracture pattern and orientation were determined from a review of injury radiographs and computed tomography scans. The incidence of subsequent mediastinal compromise was then identified via a chart review. One hundred five patients were included for analysis. Twenty-two patients (20.8%) had computed tomography scans for review. The average age was 56 years, with 53% of patients being male. Sixty-eight percent of patients reported a high-energy mechanism of injury. No patients demonstrated evidence of mediastinal compression on physical examination. No patients required hospitalization for complications secondary to mediastinal compromise. Ninety percent (n=94) of patients were treated nonoperatively. Forty-three percent of fractures were nondisplaced. The remaining fractures demonstrated anterior or superior displacement of the lateral fragment, with a 0% incidence of posterior displacement. The most common indication for surgery was fracture displacement (n=10). A classification of medial clavicle fractures was developed using data from our cohort and a literature review. Medial clavicle fractures rarely demonstrate posterior displacement. Despite fracture proximity, mediastinal injury is exceedingly uncommon. [ Orthopedics . 2023;46(3):e161-e166.].

Glenoid bone loss presents a challenging dilemma, particularly in the setting of failed arthroplasty requiring conversion to a reverse total shoulder arthroplasty (rTSA). The aim of our systematic review was to examine the success and failure of biologic glenoid bone grafting to address vault deficiencies in the setting of shoulder arthroplasty conversion to rTSA. Twelve articles were included and a complete PUBMED search. Inclusion criteria included glenoid bone grafting for conversion of failed arthroplasty and a minimum of 12 months follow-up. Exclusion criteria included grafting for primary rTSA, and re-revision for infection or humeral loosening. Failures were defined as failure of the graft to radiographically incorporate, symptomatic base plate loosening, and need for further surgical re-revision. Two hundred patients were identified across the 12 articles. Eighteen percent (36/200) of all cases demonstrated failure to radiographically incorporate. Thirteen percent (25/200) of all grafting cases required re-revision due to symptomatic failure (pain or functional deterioration). Femoral shaft demonstrated the highest failure rate at 88% (7/8). Grafting for glenoid bone loss in the setting of conversion to rTSA has an 82% rate of success across autograft and allograft utilization. Further studies are needed to better define the success of autografting versus allografting in the setting of shoulder arthroplasty conversion to rTSA with glenoid bone loss., (© 2022 Published by Elsevier Inc. on behalf of American Shoulder & Elbow Surgeons.)

Purpose: We sought to determine the effectiveness of corticosteroid injections (CSIs) for de Quervain tenosynovitis in patients with diabetes mellitus., Methods: We retrospectively identified all patients with diabetes receiving a CSI for de Quervain tenosynovitis by 16 surgeons over a 2-year period. Data collected included demographic information, medical comorbidities, number and timing of CSIs, and first dorsal compartment release. Success was defined as not undergoing an additional CSI or surgical intervention. The mixture of a corticosteroid and local anesthetic provided in each injection was at the discretion of each individual surgeon., Results: Corticosteroid injections were given to 169 wrists in 169 patients with diabetes. Out of 169 patients, 83 (49%) had success following the initial CSI, 44 (66%) following a second CSI, and 6 (67%) following a third CSI. A statistically significant difference was identified in the success rates between the first and second CSIs. Ultimately, 36 of 169 wrists (21%) underwent a first dorsal compartment release., Conclusions: Patients with diabetes mellitus have a decreased probability of success following a single CSI for de Quervain tenosynovitis in comparison to nondiabetic patients, as described in the literature. However, the effectiveness of each additional CSI does not appear to diminish., Type of Study/level of Evidence: Therapeutic IV., (Copyright © 2022 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Background: Despite surgical stabilization of complex elbow trauma, additional fixation to maintain joint congruity and stability may be required. Multiple biomechanical constructs include a static external fixator (SEF), a hinged external fixator (HEF), an internal joint stabilizer (IJS), and a hinged elbow orthosis (HEO). The optimal adjunct fixation to surgical reduction is yet to be determined., Methods: Eight matched cadaveric upper extremities were tested in a biomechanical model. Anteroposterior stress radiographs were obtained of the elbow in full supination at 0° and 45° of elbow flexion with the weight of the hand serving as a varus load as the baseline. A 360° capsuloligamentous soft-tissue release was performed around the elbow. The biomechanical constructs were applied in the same sequential order: SEF, HEF, IJS, and HEO. For each construct, 0 kg (0-lb) and 2.3 kg (5-lb) of weight were applied to the distal arm. At both weights, radiographs were obtained with the elbow at 0° and 45° of flexion, with subsequent measurement of displacement, congruence at the ulnohumeral joint, and the ulnohumeral opening angle. Statistical analysis was performed to quantify the strength and stability of each construct., Results: Compared with the control group at 0° with and without 2.3 kg (5-lb) of varus force and at 45° with and without 2.3 kg (5-lb) of varus force, no difference was noted in the medial ulnohumeral joint space, lateral ulnohumeral joint space, or ulnohumeral opening angle between the SEF, HEF, and IJS. The gap change after exertion of a 2.3-kg (5-lb) force between the control condition and application of each construct demonstrated no difference between the SEF, HEF, and IJS. Comparison among destabilized elbows showed no significant difference between the SEF, HEF, and IJS. The HEO catastrophically failed in each position at 0 kg (0-lb) of weight., Conclusion: The SEF, HEF, and IJS are neither superior nor inferior at maintaining elbow congruity with the weight of the arm and 2.3 kg (5-lb) of varus stress. The HEO did not provide additional stability to the unstable elbow., (Copyright © 2022 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Subchondral bone marrow edema (SBME) represents a pathologic alteration of subchondral bone. A strong correlation exists between its presence and the progression of osteoarthritis. Very few treatment options exist between the spectrum of conservative management and the definitive treatment of total knee arthroplasty (TKA). Tactoset® is an injectable synthetic, biocompatible hyaluronic acid-based bone graft substitute that allows for a minimally invasive treatment for painful SBME via percutaneous skeletal fixation (PSF). We present the technique of PSF using Tactoset., (© 2020 by the Arthroscopy Association of North America. Published by Elsevier.)

Purpose: To investigate the change in patient-reported pain after percutaneous skeletal fixation (PSF) and to determine the success rate of PSF in the prevention of additional intervention for the treatment of painful subchondral bone marrow edema (SBME) of the knee over a 2-year postoperative period., Methods: This was a retrospective, single-surgeon analysis of patients undergoing PSF for painful, atraumatic SBME of the knee confirmed on preoperative magnetic resonance imaging with a minimum 2-year follow-up. Inclusion criteria were age >18 years, pain localized to the area of edema, failure of nonsurgical intervention (4 weeks of physical therapy and non-steroidal medication use), and absence of tricompartmental Kellgren-Lawrence grade 4 osteoarthritis. All patients underwent arthroscopy, followed by isolated PSF without additional chondral procedures. Pre- and postoperative visual analog scale scores were compared. The primary outcome measure of success was defined as a lack of additional intervention. This included viscosupplementation, corticosteroid injection, or conversion to arthroplasty., Results: A total of 74 patients with a mean age of 47.2 years and average follow-up time of 38.9 months (range 24-61 months) were evaluated. Successful treatment was noted in 61 patients (82.4%). Of the 13 patients who did not respond to PSF, 5 (6.8%) had been converted to arthroplasty, 11 received viscosupplementation, and 8 required cortisone injections. The average visual analog scale score decreased from 7.55 preoperatively to 3.16 at 2-year follow-up ( P < .001). The average body mass index of successfully treated patients (28.2) was significantly less than that of the patients experiencing failure (32.2) ( P  = .001)., Conclusions: Patients undergoing PSF for the treatment of painful SBME may expect a decrease in knee pain and low rates of additional intervention over a 2-year postoperative period., Level of Evidence: Level IV; Therapeutic Case Series., (© 2020 by the Arthroscopy Association of North America. Published by Elsevier Inc.)

Purpose: To determine whether deleted in colon cancer (DCC) protein expression in colorectal cancer (CRC) metastases could predict outcome to palliative fluorouracil (FU)-based chemotherapy and to assess whether it is similar to that observed in the corresponding primary tumors., Patients and Methods: DCC protein expression was assessed immunohistochemically on archival specimens of CRC metastases from 42 patients homogeneously treated by methotrexate-modulated bolus FU alternated to 6-S-leucovorin-modulated infused FU and was retrospectively correlated with patient characteristics and clinical outcome. In a subset analysis, DCC immunoreactivity was compared between metastatic CRC and the corresponding primary tumors and regional lymph node metastases., Results: Positive immunoreactivity for DCC was found in 45% of patients. Eighteen (78%) of 23 patients for whom multiple samples were available displayed a similar pattern of expression in distant metastases and primary tumors. The median survival time was 14.3 months in patients without DCC expression and 21.4 months in patients with DCC-positive tumors (log-rank test, P =.04); the 2-year survival rates were 8.5% and 42.5%, respectively. Response rates to chemotherapy were not significantly different between the two groups. By multivariate analysis, DCC protein expression maintained its prognostic value and showed to be the single best predictor of survival, with a relative risk of 2.16., Conclusion: Our results indicate that expression of the DCC protein in CRC metastases is similar to that observed in the corresponding primary tumors and represents a dominant predictor of survival in patients with unresectable, advanced CRC who are undergoing palliative FU-based chemotherapy.

Background: Different 5-fluorouracil (5-FU) schedules and/or biochemical modulators may result in different mechanisms of cytotoxicity, potentially affecting the correlation between thymidylate synthase (TS) expression and the clinical response to the fluoropyrimidine., Patients and Methods: TS levels were measured immunohistochemically on archival specimens of colorectal cancer metastases from 124 patients homogeneously treated in a series of clinical trials at our institutions with: (A) leucovorin (LV)-modulated infusional 5-FU (n = 48); (B) LV-modulated bolus 5-FU (n = 41); (C) methotrexate (MTX)-modulated bolus 5-FU (n = 35)., Results: A statistically significant correlation between TS levels and the clinical response was observed with the regimens involving continuous infusion and/or LV modulation (response rate in patients with low and high TS: 66% versus 24%, P = 0.003, and 50% versus 0%, P = 0.0001, in group A and B, respectively). Conversely, TS levels failed to predict the clinical response within the group of patients treated with MTX-modulated bolus 5-FU (response rate 21% versus 13%, P = 0.50, with low and high TS, respectively). Consistently, the median time to progression/overall survival time in patients with low and high TS were 9 versus 6 months/19 versus 14 months (P = 0.009/0.035, group A), 8 versus 2 months/12 versus 6 months (P = 0.002/0.0006, group B) and 3 versus 2 months/12 versus 13 months (P = 0.14/0.74, group C)., Conclusions: The correlation between intratumoral TS levels and the clinical response to 5-FU depends strongly on the schedule of administration/biochemical modulators that are used in different 5-FU regimens. These data strengthen the notion that different 5-FU schedules have different mechanisms of cytotoxicity.

Thymidylate synthase (TS) expression in colorectal cancer metastases has been shown to predict for the clinical response to 5-fluorouracil. Because primary tumors may easily provide accessible sources of tissue for marker analysis, we have investigated the stability of TS expression between primary colorectal cancer and the corresponding distant metastases and compared their relative ability to predict response to chemotherapy on a series of 27 patients homogeneously treated with biochemically modulated fluorouracil for advanced disease. By immunohistochemistry, high levels of TS expression were observed in 19 of 27 (70%) primary tumors and in 13 of 27 (48%) metastatic samples. Overall, TS levels observed in primary tumors did not correlate with those measured in the corresponding metastases (r = 0.30, P = 0.13), with higher TS levels in primary tumors in 8 of 10 discordant cases. Accordingly, the degree of TS immunoreactivity was significantly higher in primary tumors compared with the corresponding metastases (mean TS score 3.8; median, 4 versus 2.8; median 3; P = 0.001). Response rates after chemotherapy for metastatic disease were similar for patients with low and high TS levels in their primary tumors (37% versus 53%, P = 0.47). In contrast, response rates were 71% and 23% in patients with low and high TS in metastatic samples (P = 0.012), respectively. In summary, TS levels measured in primary colorectal cancer do not reflect those observed in the corresponding metastases and cannot be used to predict their response to chemotherapy. The basis for the higher TS content of primary colorectal cancer compared with the corresponding metastases needs clarification.

Background: We assessed the hypothesis that a compromised p53 function could account for the non response of colon cancer patients with low thymidylate synthase (TS) expression receiving a bolus 5-fluorouracil (5-FU) leucovorin (LV) combination., Patients and Methods: The study population consisted of 41 patients with unresectable metastatic colon cancer, homogeneously, treated with bolus 5-FU and LV., Results: Twenty-seven patients (66%) showed high levels of TS expression. The difference in the proportion of objective responses between patients with low (CR + PR: 7 of 14, 50%) and high (CR + PR: 0 of 27) TS levels was statistically significant (P = 0.0001, chi-square test). p53 nuclear over-expression was found in 27 of 41 patients (66%). No differences were observed in p53 overexpression in patients with high (66%) or low (66%) TS expression. p53 status was not found to be associated with response even in patients with low TS expression., Conclusions: p53 status measured by immunohistochemistry does not seem to be useful to identify unresponsive patients with low TS expression.

Recently, we have demonstrated that thymidylate synthase (TS) protein expression predicts for the clinical response to a regimen of infusional 5-fluorouracil (5FU) in advanced colorectal cancer patients. Previous studies by other groups that showed a correlation between TS gene expression and response to the fluoropyrimidine also involved infusional regimens. Considering the putatively different mechanism of action of bolus compared with continuous infusion of 5FU, the aim of the present study was to test whether the correlation between TS expression and the clinical response to 5FU is valid for bolus regimens. A secondary aim was to compare TS levels between liver metastases and abdominal recurrences from colon cancer, because these sites have a distinctly different responsiveness to 5FU chemotherapy. The study population consisted of 41 patients (25 males and 16 females; median age, 60 years) with unresectable metastatic or recurrent colon cancer, homogeneously treated with 5FU (420 mg/m2 i.v., days 1-5) and leucovorin (20 mg/m2 i.v., days 1-5); cycles were repeated every 28 days. Twenty-seven patients (66%) showed high levels of TS expression as defined by TS scores equal to 3 and 4. The proportion of cases with high levels of TS expression was significantly higher in abdominal recurrences (18 of 22, 82%) compared with liver metastases (9 of 19, 47%; P = 0.02). Intratumoral TS protein expression was inversely correlated with response to chemotherapy (response rate: 7 of 14, 50%, versus 0 of 27 in patients with low and high levels of TS expression, respectively; P = 0.0001). These results confirm that the level of TS protein expression predicts for response to 5FU, even with a bolus schedule. The higher TS levels observed in abdominal compared with liver metastases may account for their different responsiveness to 5FU chemotherapy. Immunohistochemical quantitation of TS protein levels may thus allow us to change the therapeutic approach to advanced colorectal cancer from a general to an individual treatment strategy at a time when new non TS-targeted drugs have become available for this disease.

Purpose: To determine whether immunohistochemical thymidylate synthase (TS) quantitation predicts for clinical outcome in patients with advanced colorectal cancer treated by fluorouracil (FUra)-based chemotherapy., Patients and Methods: TS levels were measured immunohistochemically on archival specimens of colorectal cancer metastases from 48 patients homogenously treated by bolus FUra plus methotrexate alternating with continuous-infusion FUra plus leucovorin. These measurements were retrospectively correlated with patient characteristics and clinical outcome., Results: A significant correlation was found between intratumoral TS expression and all the parameters of clinical outcome analyzed. In patients whose tumors had low (n = 27) and high (n = 21) TS levels, the overall response rates were 67% and 24%, respectively (P =.003). The percentage of tumor shrinkage after chemotherapy was linearly related to TS immunoreactivity (r =.56, P =.00004), and its mean values were 65% and 14% with low and high TS levels, respectively (P =.0001). By logistic regression analysis, low TS expression was the single best predictor of response to chemotherapy (relative probability, 5.0). In patients with low and high TS expression, the median time to progression was 9.6 months v 6.2 months (P =.005) and the median survival time 18.4 months v 15.4 months (P =.02), respectively. Two- and 3-year survival rates were 41% v 15% and 19% v 0% (P =.02), respectively., Conclusion: In this cohort of homogenously treated patients, intratumor TS content was a major predictor of clinical outcome. Immunohistochemical TS quantitation provides a convenient, low-cost technique for identifying patients unresponsive to TS inhibitors who may be candidates for alternative chemotherapy regimens.

The quinazoline folate analogue raltitrexed (ZD1694; Tomudex) and the camptothecin derivative irinotecan are two new agents showing clinical activity against colorectal cancer. To identify the optimal conditions to achieve synergistic cytotoxicity before the clinical development of their combination, we explored the interactions between ZD1694 and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), in vitro. Cytotoxicity was evaluated with a clonogenic assay using the human colon cancer cell line HCT-8. Different schedules of administration and different dose ratios of the two agents were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27-55, 1984). Sequential short-term (1 and 4-h) exposures to SN-38 followed by ZD1694 resulted in synergistic cytotoxicity at broad dose-effect ranges. At a high level of cell kill, the synergism was greater when either equiactive doses of the two agents or higher relative doses of ZD1694 were used. A 24-h interval between exposure to SN-38 and ZD1694 significantly enhanced the magnitude of the synergy (P = 0.001). The opposite sequence or simultaneous exposures produced significantly less potentiation or nearly additive interactions (P = 0.0006 and P < 0.0001). The synergism was completely lost under conditions of more prolonged drug exposure (24-h continuous exposure). In conclusion, in this in vitro model of human colon cancer, ZD1694 and SN-38 produced synergistic cytotoxicity. Our findings support the clinical use of this combination and provide a rationale for a clinical trial using sequential short-term exposures to equiactive doses of SN-38 and ZD1694 administered sequentially with a 24-h interval.

Nine human ovarian cancer cell lines that express wild-type (wt) or mutated p53 were used to evaluate the cytotoxicity induced by paclitaxel. The IC50 calculated in the five mutated p53-expressing cell lines was not different from the four wt p53-expressing cell lines. The introduction of wt p53, by using a temperature-sensitive mutant murine p53 or the human p53 under the control of a tetracycline-dependent promoter, did not change the cytotoxicity of paclitaxel as compared to mock-transfected cells. By using for each cell line the paclitaxel IC50, we found that these concentrations were sufficient to induce an increase in p53 levels in all of the four wt p53-expressing cells, whereas in the mutated p53-expressing cells, the levels were unaffected. This increase in p53 levels led to an increase in the mRNA and protein levels of p53 downstream genes (WAF1, GADD45, and bax). In none of the cell lines examined was paclitaxel able to induce apoptosis, evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling staining and filter binding assay at concentrations closed to the IC50. By increasing the concentration of paclitaxel in the filter binding assay, we could see fragmentation of DNA in the different cell lines. We conclude that the presence of p53 is not a determinant for the cytotoxicity induced by paclitaxel in human ovarian cancer cell lines. Differences in the activation of p53 downstream genes could be observed in wt versus mutated p53-expressing cells, but this does not account either for a differential induction of apoptosis or for a change in cytotoxicity induced by paclitaxel.

Nine human ovarian cancer cell lines that express wild-type (wt) or mutated (mut) p53 were used to evaluate the cytotoxicity induced by cisplatin (DDP). The concentrations inhibiting the growth by 50% (IC50) were calculated for each cell line, and no differences were found between cells expressing wt p53 and mut p53. Using, for each cell line, the DDP IC50, we found that these concentrations were able to induce an increase in p53 levels in all four wt-p53-expressing cell lines and in one out of five mut-p53-expressing cell lines. WAF1 and GADD45 mRNAs were also increased by DDP treatment, independently of the presence of a wt p53. Bax levels were only marginally affected by DDP, and this was observed in both wt-p53- and mut-p53-expressing cells. DDP-induced apoptosis was evident 72 h after treatment, and the percentage of cells undergoing apoptosis was slightly higher for wt-p53-expressing cells. However, at doses near the IC50, the percentage of apoptotic cells was less than 20% in all the cell lines investigated. We conclude that the presence of wt p53 is not a determinant for the cytotoxicity induced by DDP in human ovarian cancer cell lines.

In this study, we demonstrated that tumor necrosis factor (TNF), secreted endogenously by four human ovarian cancer cell lines (A2774, IGROV-1, OVCAR-8, SW626), is biologically active against L929 cells and its activity is specifically inhibited by anti-TNF antibodies. Its endogenous production is increased by treatment for 24 h with phorbol myristate acetate (PMA)/ Ionomycin (Iono). All cell lines express TNF high-affinity receptors and release only 60-kdalton soluble TNF receptor, both spontaneously and after stimulation with PMA/Iono. TNF endogenously secreted by human ovarian cancer cell lines is very efficient in potentiating the activity of DNA topoisomerase II inhibitors (doxorubicin, mitoxantrone, VP16). The activity of vinblastine and bleomycin is not potentiated and, more interestingly, cisplatin's activity is inhibited. In 24-h PMA/Iono-stimulated A2774 cells, mitoxantrone specifically generated more cleavable complexes than in unstimulated cells. This result could provide an important tool in the therapy of human ovarian cancer secreting TNF protein, previously considered as a negative prognostic factor.

Taxol is an antitumor drug which, as its mechanism of action, promotes microtubule assembly in vitro. Camptothecin (CPT) is an anticancer agent with the peculiar mechanism of poisoning eukaryotic DNA topoisomerase I. Both drugs are in clinical trials and their chemotherapeutic efficacy seems promising in refractory human ovarian cancer. We studied the molecular and cellular pharmacology of the two drugs when administered simultaneously toward human ovarian cancer cell line A2780. Taxol inhibits CPT-induced single-strand breaks as well as CPT-induced cytotoxicity. Taken together, our experiments indicate that the two drugs might interact with the same class of nuclear enzyme, i.e. DNA topoisomerase I.

The 'nicotinamide adenine dinucleotide phosphate diaphorase' histochemical technique was used as a marker of neuronal nitric oxide synthase to assess the presence of the enzyme in the anterior hypothalamus of the rat. Particular attention was focused on the subparaventricular zone, periventricular area and suprachiasmatic nucleus. The results show that there is strong staining in the anterior hypothalamus particularly in the subparaventricular zone by the perinuclear regions of the suprachiasmatic nucleus, and in the periventricular nucleus. Some diaphorase activity was also seen within the suprachiasmatic nucleus, but this was much weaker than in the surrounding areas. These results, taken together with existing evidence, would further suggest the involvement of nitric oxide in the signal transduction pathway in the suprachiasmatic nucleus.

The combination of 5-fluorouracil (FUra) plus IFN-beta was studied in vitro using a human colon carcinoma cell line, HCT-8. Continuous exposure to high concentrations of IFN-beta is cytotoxic to these cells (ED50, 600 +/- 50 IU/ml). A strong synergism (P < 0.002) was observed when a short-term (1-h), high-concentration exposure to fluoropyrimidine (300 or 1000 microM) was followed by IFN-beta given continuously. In fact, the mean ratio between the expected (product of the survival of each agent alone) and the observed clonogenic cell survival rates of the combination was 3.4 (range, 2.4-4.9). Longer exposures to the fluoropyrimidine (24 h, 7 days) produced less than additive effects with IFN-beta, indicating strong schedule dependency for this synergism. The mechanism of interaction was studied at four levels. First, thymidylate synthase (TS) activity, inhibition, and recovery were measured by an in situ assay in cells treated with FUra, IFN-beta, and their combination. When the prolonged infusion of IFN-beta followed a 1-h exposure to FUra, the observed TS inhibition and recovery, at each time point, were very similar to the expected values, indicating that the interactions between these drugs at the level of TS are not the determinant of the synergism. Second, cell cycle analysis was done. During the continuous exposure to IFN-beta, a significant accumulation of HCT-8 cells in S-phase was observed at 24, 48, and 72 h compared to untreated controls (P = 0.003); however, under the same experimental conditions producing synergy in the clonogenic assay, no significant cell cycle perturbations were produced by the combination of FUra followed by IFN-beta compared to those caused by each agent alone. Third, using the alkaline elution test, we also demonstrated that the synergism is not due to enhanced FUra-induced DNA single-strand break frequency in high molecular weight DNA. Finally, nucleic acid incorporation experiments, using tritiated FUra, showed that the cytokine, given continuously (300 IU/ml), enhanced the amount of FUra incorporated into nucleic acids 24 h after a 1-h exposure to 300 and 1000 microM of FUra. The median percentage of enhancement values were 31.6 +/- 11.5%,m for the lower drug concentration and 18. 4 +/- 8.1% for the higher drug concentration tested. These results suggest that the mechanism of this synergism may be related to the ability of IFN-beta to promote the incorporation of intracellular FUra metabolites into nucleic acids and/or to inhibit the cleavage of FUra nucleotides from RNA/DNA.

The effect of interferon-alpha or beta on platinum analogues [cisplatin (CDDP) and carboplatin] cytotoxicity was studied in four glioblastoma cell lines (U373MG, T98G, A172 and U118Mg). All cell lines were strongly resistant to the cytotoxic effect of CDDP or carboplatin. Although both interferons were not cytotoxic in all cell lines, they were able to significantly increase the cell platinum-sensitivity. Specifically interferon-alpha increased the magnitude of CDDP-induced DNA interstrand crosslinks. Our findings suggest that interferons are able to induce a very strong potentiation of platinum analogues cytotoxicity in drug-resistant human glioma cell lines.

Seven ovarian and one cervical human cancer cell lines were examined far their sensitivity or resistance to tumor necrosis factor, to three topoisomerase II inhibitors and to cisplatin. Only one line exhibited the multidrug-resistance phenotype and another one an 'atypical'-MDR phenotype. The combination of TNF and topoisomerase-II inhibitors produced enhanced cytotoxicity and overcame the MDR and the atypical resistance. No potentiation of cisplatin cytotoxicity was observed. These findings suggest that TNF enhances the activity of DNA topoisomerase II both in TNF resistant and sensitive cells.