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Sixty-five patients with recurrent aphthous stomatitis participated in a double-blind study to evaluate levamisole. Nineteen of 34 patients improved on levamisole and only five of 31 patients improved on placebo (P<0.004, chi-square with two degrees of freedom). After breaking the code and switching placebo patients to levamisole and non-responders to an alternate dosage schedule, 46 of 60 patients noted clinical improvements. [ABSTRACT FROM AUTHOR]

Background: Routine sentinel lymphadenectomy (SLNB) for early-stage HR+/HER2- breast cancer in women ≥70 is discouraged by Choosing Wisely, but whether SLNB can be routinely omitted in women ≥70 with DCIS undergoing mastectomy is unclear. This study aims to evaluate rates of axillary surgery and nodal positivity (pN+) in this population to determine the impact of axillary surgery on treatment decisions., Methods: Females ≥70 with DCIS undergoing mastectomy were identified from the National Cancer Database (2012-2020). The rate of upstaging to invasive cancer (≥pT1) or pN+ was assessed. Subset analyses were conducted for ER+ patients. Adjuvant therapies were evaluated among ≥pT1 patients after stratifying by nodal status., Results: Of 9,030 patients, 1,896 (21%) upstaged to ≥pT1. Axillary surgery was performed in 86% of patients, predominantly sentinel lymphadenectomy (SLNB, 65%). Post hoc application of Choosing Wisely criteria demonstrated that 93% of the entire cohort and 97% of ER+ DCIS patients could have avoided axillary surgery. Nodal positivity was 0.3% among those who didn't upstage, and 12% among those upstaging to ≥pT1, with <2% having pN2-3 disease, irrespective of receptor subtype. Node-positive patients had higher adjuvant therapy usage, but there was no recommendation for adjuvant chemotherapy or radiation for 71% and 66% of pN+ patients, respectively., Conclusions: Axillary surgery can be omitted for most patients ≥70 undergoing mastectomy for ER+ DCIS, aligning with recommendations for invasive cancer, and omission can be considered in those with ER- disease. Future guidelines incorporating preoperative imaging, as in the SOUND trial, may aid in identifying patients benefiting from axillary surgery., (© 2024. Society of Surgical Oncology.)

Background: Surgical stabilization of rib fractures (SSRF) and surgical stabilization of sternal fractures (SSSF) involves open reduction and internal fixation of fractures with an implantable titanium plate to restore and maintain anatomic alignment. The presence of this foreign, non-absorbable material presents an opportunity for infection. Although surgical site infection (SSI) and implant infection rates after SSRF and SSSF are low, they present a challenging clinical entity. Methods: The Surgical Infection Society's Therapeutics and Guidelines Committee and Chest Wall Injury Society's Publication Committee convened to develop recommendations for management of SSIs or implant-related infections after SSRF or SSSF. PubMed, Embase, Web of Science and the Cochrane database were searched for pertinent studies. Using a process of iterative consensus, all committee members voted to accept or reject each recommendation. Results: For patients undergoing SSRF or SSSF who develop an SSI or an implant-related infection, there is insufficient evidence to suggest a single optimal management strategy. For patients with an SSI, systemic antibiotic therapy, local wound debridement, and vacuum-assisted closure have been used in isolation or combination. For patients with an implant-related infection, initial implant removal with or without systemic antibiotic therapy, systemic antibiotic therapy with local wound drainage, and systemic antibiotic therapy with local antibiotic therapy have been documented. For patients who do not undergo initial implant removal, 68% ultimately require implant removal to achieve source control. Conclusions: Insufficient evidence precludes the ability to recommend guidelines for the treatment of SSI or implant-related infection following SSRF or SSSF. Further studies should be performed to identify the optimal management strategy in this population.

Purpose: The use of metallic implants for the treatment of patella fractures often involves complications related to the hardware. As a result, there has been a considerable focus on the use of non-metallic implants. The aim of this study was to determine the differences in clinical outcomes, including the rates of complications and re-operations following the surgical management of patellar fractures with metallic versus non-metallic implants., Methods: The systematic review was conducted in accordance with PRISMA guidelines. Two investigators independently applied the search strategy to PubMed, SCOPUS, Google Scholar, and Cochrane databases, limited to publications between 1st January 2000 to 30st September 2021. The search strategy identified studies in which there was a comparison between the clinical outcome of the metallic and non-metallic fixation. Meta-analysis was conducted according to the Cochrane Collaboration and Quality of Reporting of Meta-Analysis (QUORUM) guidelines., Results: A total of 19 studies were selected for the systematic review and seven studies for the meta-analysis. Two hundred ninety-sixt patients formed the pooled study population for the meta-analysis. The outcomes evaluated in the meta-analysis were range of motion (ROM), complications, and re-operation rates. There was no significant difference between groups regarding ROM, but the risk ratio (RR) of re-operation (RR 0.19, 95% CI [0.09-0.41]) and complications (RR 0.30, 95% CI [0.17-0.55]) was significantly in favor of fixation with non-metallic implants., Conclusion: The use of non-metallic implants for fixation of patella fractures was associated with significantly lower risk of complications and re-operations than fixation with metallic implants, without any difference between groups with respect to the final range of motion or functional outcome measures., (© 2022. The Author(s) under exclusive licence to SICOT aisbl.)

Objective: Ultrasound provides accessible imaging for bedside diagnostics and procedural guidance, but may lead to misdiagnosis in untrained users. The main objective of this study was to determine observed and self-perceived competence with critical care ultrasound in graduated general surgery residents., Design: The design of this study was a retrospective review. Ultrasound training program records were reviewed for number of prior ultrasound examinations performed, self-perceived competence, observed competence on faculty examinations, and intended future use of individual critical care ultrasound examinations., Setting: This study was undertaken at the R Adams Cowley Shock Trauma Center, which is a tertiary care center in Baltimore, MD., Participants: Graduated general surgery residents were identified at the beginning of their surgical critical care fellowship at our institution, and were included if they participated in our critical care ultrasound education program. Fifteen graduated general surgery residents were included., Results: Prior ultrasound experience ranged from 100% for focused assessment of sonography for trauma (FAST) to 13.3% for advanced cardiac assessment. Self-perceived competence ranged from 46.7% with FAST to 0% for advanced cardiac assessment. Observed competence ranged from 20.0% for FAST examinations to 0% for basic cardiac assessment, advanced cardiac assessment, and inferior vena cava (IVC) assessment. All participants intended to use ultrasound in the future for FAST, pneumothorax detection and basic cardiac assessment, and 86.7% for IVC assessment and advanced cardiac assessment. Of participants with self-perceived competence, 28.6% had observed competence with FAST, 0% with IVC assessment, and 100% with pneumothorax detection., Conclusions: Graduated general surgery residents are not competent in multiple critical care ultrasound examinations despite universally planning to use critical care ultrasound in future practice. Current exposure to ultrasound in residency may give a false sense of competency with ultrasound use. A standardized ultrasound curriculum is an urgent need for general surgery training., (Published by Elsevier Inc.)

Objectives: Missed pancreatic injury carries significant morbidity. Computerized tomography (CT) imaging is useful, but may lack sensitivity to identify pancreatic injury. New-generation CT scanners should improve sensitivity, but this has not been studied. A previous study published in 2002 evaluating the sensitivity for identifying pancreatic injury with single-slice CT scanners yielded a 68% correlation between operative and CT findings. We aim to study the accuracy of modern CT for diagnosis and grading of pancreatic injury., Methods: All trauma admissions from 2008 and 2012 were retrospectively reviewed. Patients with a pancreatic injury, either on CT or intraoperatively, were included (n = 96). Sensitivity and specificity were calculated using Student's t test., Results: 48 patients had injuries noted on CT and in the operating room. In this group, 68.8% had CT findings discordant with operative findings. Of these, 78.8% had no injury noted on CT, of which 26.9% required surgical intervention. Seven patients with injury on CT had none identified in the operating room. Based on these results, the sensitivity for CT imaging to identify an injury is 36.4% with a positive predictive value of 68.2%., Conclusions: Our results indicate that despite advances in CT technology, the sensitivity and specificity for identifying pancreatic injury remains low. Although CT scans remain critical in trauma evaluation, awareness of this diagnostic gap is important. Further analysis is required to determine any impact on patient outcomes., (Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.)

Ameloblastic carcinoma (AC) is an uncommon malignant odontogenic tumor that can be difficult to differentiate from ameloblastoma and can arise directly as an undifferentiated lesion or from a pre-existing benign lesion. The current study presents a novel case of primary maxillary AC and review the literature on AC of the maxilla. The review of the literature indicates that secondary tumors and posterior localization are associated with a higher tendency for recurrence and, often, multiple recurrences. Surgical therapy, eventually followed by radiotherapy, is the treatment modality most frequently applied, while the role of chemotherapy remains unclear. Several new cases of maxillary AC have been recently described in literature, making this pathology more frequent than previously considered; this is perhaps an indication of an increased diagnostic sensibility, rather than a real increase in the incidence of the disease itself.

Background: Point-of-care ultrasound often includes cardiac ultrasound. It is commonly used to evaluate cardiac function in critically ill patients but lacks the specific quantitative anatomic assessment afforded by standard transthoracic echocardiography (TTE). We developed the Focused Rapid Echocardiographic Examination (FREE), a hybrid between a cardiac ultrasound and TTE that places an emphasis on cardiac function rather than anatomy. We hypothesized that data obtained from FREE correlate well with TTE while providing actionable information for clinical decision making., Methods: FREE examinations evaluating cardiac function (left ventricular ejection fraction), diastolic dysfunction (including early mitral Doppler flow [E] and early mitral tissue Doppler [E']), right ventricular function, cardiac output, preload (left ventricular internal dimension end diastole), stroke volume, stroke volume variation, inferior vena cava diameter, and inferior vena cava collapse were performed. Patients who underwent both a TTE and FREE on the same day were identified as the cohort, and quantitative measurements were compared. Correlation analyses were performed to assess levels of agreement., Results: A total of 462 FREE examinations were performed, in which 69 patients had both a FREE and TTE. FREE ejection fraction was strongly correlated with TTE (r = 0.89, 95% confidence interval). Left ventricular outflow tract, left ventricular internal dimension end diastole, E, and lateral E' derived from FREE were also strongly correlated with TTE measurements (r = 0.83, r = 0.94, r = 0.77, and r = 0.88, respectively). In 82% of the patients, right ventricular function for FREE was the same as that reported for TTE; pericardial effusion was detected on both examinations in 94% of the cases. No significant valvular anatomy was missed with the FREE examination., Conclusion: Functionally rather than anatomically based hybrid ultrasound examinations, like the FREE, facilitate decision making for critically ill patients. The FREE's functional assessment correlates well with TTE measurements and may be of significant clinical value in critically ill patients, especially when used in remote operating environments where resources are limited., Level of Evidence: Diagnostic test, level III.

Background: Early diagnosis and treatment of traumatic brain injury (TBI) lead to better outcomes. It is difficult to predict which patients benefit from specialised centres, leading to over triage or delay in definitive care. We propose that a non-invasive test comprising optic nerve sheath ultrasound, transcranial Doppler and quantitative papillary reactivity is feasible, correlates with CT findings and may allow for accurate early identification of TBI., Methods: A 1-year, prospective observation study evaluated a low-risk, non-invasive method of assessing brain injury. Patients underwent a non-invasive neurological examination for trauma, including the above assessments. Data from the three examinations were collected within 6 hours of injury and at 24 hours, and were analysed. Demographics, haemodynamic data, imaging results and short-term outcomes/interventions were recorded., Results: Trauma patients over the age of 18 years, with a Glascow coma scale (GCS) of <12 or CT evidence of TBI, and intubated were included (N=100). These were divided into +CT (n=49) and -CT groups (n=51) according to the Marshall CT classification of TBI. The +CT group was older, with worse GCS and higher lactate (p=0.008, p=0.001 and p=0.01) but were otherwise well matched. The +CT group included all TBI types, with 96% of the patients having more than one type of TBI. Pulsatility index and neurologic pupillary index were predictive of a +CT (p=0.04, p=0.02). Area under the receiver-operating curve for the logistic regression model for the prediction of positive radiographic findings was r=0.718. Finally, we suggest a preliminary scoring heuristic for predicting a positive radiological finding in a patient with TBI., Conclusions: The proposed examination is a feasible, non-invasive tool that may have clinical utility in the early prediction of TBI. If validated, it may improve trauma triage for the brain-injured patient. Further studies are warranted to validate this model., Competing Interests: Competing interests: The authors have no disclosures or conflict of interest to report. JG, MV and CC are Active Duty Military Surgeons. Reference to any product does not constitute nor imply its endorsement, recommendation or favouring by the USA government or any agency thereof. The views and opinions of the authors do not necessarily state nor reflect those of the USA government.

Objectives: To determine the specific CT findings of penetrating neck wound profile predicting aerodigestive injuries, diagnostic performance of CTA and to propose a modified selective management algorithm to reduce nontherapeutic invasive procedures., Methods: We retrospectively evaluated CTAs of 102 patients to determine the presence of various CT signs. "Trajectory"-based signs included trajectory of the wound extending into the aerodigestive tract and trajectory violating the deep neck spaces. "Conventional" signs included transcervical injury; wall defect; air or blood in the deep neck spaces; irregular or thickened aerodigestive tract; and active mucosal bleeding., Results: Trajectory of the wound extending into the aerodigestive tract (sensitivity 76 %, specificity 97 %) and trajectory of the wound violating the suprahyoid deep neck spaces or the infrahyoid visceral space (sensitivity 97 %, specificity 55 %) were the best predictors of injury on regression analysis. The most specific "conventional" CT signs were "wall defect" and "active mucosal bleed", but had very low sensitivity. The sensitivity of CTA for detecting an injury ranged from 89.5 % to 92 %, specificity ranged from 62.5 % to 89 %., Conclusion: CTA can be a useful technique in detecting aerodigestive injury. Our proposed management algorithm can exclude an injury with high degree of confidence (sensitivity 97 %)., Key Points: • Trajectory-based CT signs predict aerodigestive injury after penetrating neck trauma. • Surgery should be considered when trajectory extends into the infra-arytenoid aerodigestive tract. • Endoscopy or exploration should be considered when trajectory violates deep neck spaces. • This modified approach can decrease negative explorations and invasive diagnostic procedures.

Background: Combat casualties have endured an increase in traumatic lower extremity amputations secondary to improvised explosive devices. Often surgical control of the proximal vasculature is required. We evaluate the safety profile of exploratory laparotomy (EXLAP) for proximal control (PC) in combat-injured patients., Methods: Records of 845 combat casualties from June 2009 to December 2011 were reviewed. Patients undergoing EXLAP were divided by indication into PC and non-PC groups. Demographics, Injury Severity Score, mechanism of injury, transfusion requirements, EXLAP findings, reoperation rates, and abdominal-related complications were recorded., Results: 44 patients were identified as PC and 91 as non-PC. Age was similar (23.7 ± 4.1 vs. 24.0 ± 4.6, p = 0.7138), and all were male. Improvised explosive devices blast was the most common mechanism of injury. Injury Severity Score (25.8 ± 8.2 vs. 21.4 ± 9.1, p = 0.0075), lower extremity amputation (93.1% vs. 28.6%, p = 0.0001), and transfusion requirements were different. Days to fascial closure (1.8 ± 1.9 vs. 1.7 ± 2.8, p = 0.8308) and number of EXLAPs were similar (2.4 ± 1.3 vs. 2.1 ± 1.5, p = 0.2581). PC had higher complications (43.1% vs. 24.2%, p = 0.0292)., Conclusion: PC demonstrated an increase in abdominal complications. The reason for this remains unclear. Alternative approaches of achieving proximal vascular control may avoid the morbidity associated with laparotomy, and predeployment training of such procedures should be considered for the general surgeon. Further studies are warranted to determine best practices for these patients., (Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.)

Background: Operation Iraqi Freedom and Operation Enduring Freedom have seen the highest rates of combat casualties since Vietnam. These casualties often require massive transfusion (MT) and immediate surgical attention to control hemorrhage. Clinical practice guidelines dictate ratio-driven resuscitation (RDR) for patients requiring MT. With the transition from crystalloid to blood product resuscitation, we have seen fewer open abdomens in combat casualties. We sought to determine the effect RDR has on achieving early definitive abdominal fascial closure in combat casualties undergoing exploratory laparotomy., Methods: Records of 1,977 combat casualties admitted to a single US military hospital from April 2003 to December 2011 were reviewed. Patients receiving an MT and laparotomy in theater constituted the study cohort. The cohort was divided into RDR, defined as a ratio of 0.8-U to 1.2-U packed red blood cells to 1-U fresh frozen plasma, and No-RDR groups. Age, injury patterns, mechanism of injury, injury severity, blood products, number of laparotomies, and days to fascial closure were collected. Assessed outcomes were number of days (early ≤ 2 days) and number of laparotomies to achieve fascial closure., Results: The mean age of the study cohort (n = 172) was 24.0 years, and mean Injury Severity Score (ISS) was 24.8. Improvised explosive device blast was the most common mechanism of injury (74.4%). The cohort was divided into RDR patients (n = 73) and no RDR (n = 99). There was no difference in mean age, mean ISS, or rate of nontherapeutic exploratory laparotomies between the groups. RDR patients had a significantly lower abdominal injury rate (34.2% vs. 72.7%, p < 0.01), had fewer laparotomies (2.7 vs. 4.3, p = 0.003), and achieved primary fascial closure faster (2.4 days vs. 7.2 days, p = 0.004). On multivariate analysis, RDR (2.74; 95% confidence interval, 1.44-5.2) was an independent predictor for early fascial closure., Conclusion: Adherence to RDR guidelines resulted in significantly decreased number of abdominal operations and was identified as an independent predictor for early fascial closure. Further investigation is warranted to validate these findings., Level of Evidence: Therapeutic study, level III.

Background: This study is a pilot experience aiming to investigate the compliance of an institutional cohort of Italian children treated for a malignant disease and their families in completing the health utilities index2, (HUI2) and the effectiveness of this measured in terms of their health status (HS) and health-related quality of life (HRQL). It specifically, it aimed to compare the HS and the HRQL, as expressed by the HUI2 global utility score, in cohorts of patients who had brain tumors, extra-cerebral solid tumors, or leukemia/lymphoma., Procedure: Fifty survivors of brain tumors, between 8 and 30 years at the time of the assessment ("self") and/or their parents ("proxy"), attending the Pediatric Oncology Out Patient clinic of Padua, Italy, completed the HUI2 questionnaire. Eighty-nine children with acute leukemia/lymphoma and 74 with extra-cerebral solid tumors and/or their parents were also assessed., Results: The mean "self" and "proxy" HUI2 global utility scores in the brain tumor patients were 0.87 and 0.84, respectively, while in the cohorts of children with other solid tumors and leukemia/lymphoma, there were 0.94, 0.91, 0.96, and 0.92, respectively. The differences between the HUI2 global utility scores in the "self" and "proxy" assessment within each cohort of children were not statistically significant. In decreasing order of frequency, the attributes affected most commonly were: "emotion," "pain," "sensation," and "cognition" both by "self" and "proxy" assessment., Conclusions: In this Italian population of childhood cancer survivors the HUI2 questionnaire proved to be a user-friendly tool, which provided information regarding HS and HRQL. A larger cohort of cancer children is needed to confirm the efficacy of the HUI2 questionnaire in distinguishing groups of children on this basis by disease category.

ATP-binding cassette (ABC) proteins include the best known mediators of resistance to anticancer drugs. In particular, ABCB1 [MDR1/P-glycoprotein (P-gp)] extrudes many types of drugs from cancer cells, thereby conferring resistance to those agents. Attempts to overcome P-gp-mediated drug resistance using specific inhibitors of P-gp has had limited success and has faced many therapeutic challenges. As an alternative approach to using P-gp inhibitors, we characterize a thiosemicarbazone derivative (NSC73306) identified in a generic screen as a compound that exploits, rather than suppresses, P-gp function to induce cytotoxicity. Cytotoxic activity of NSC73306 was evaluated in vitro using human epidermoid, ovarian, and colon cancer cell lines expressing various levels of P-gp. Our findings suggest that cells become hypersensitive to NSC73306 in proportion to the increased P-gp function and multidrug resistance (MDR). Abrogation of both sensitivity to NSC73306 and resistance to P-gp substrate anticancer agents occurred with specific inhibition of P-gp function using either a P-gp inhibitor (PSC833, XR9576) or RNA interference, suggesting that cytotoxicity was linked to MDR1 function, not to other, nonspecific factors arising during the generation of resistant or transfected cells. Molecular characterization of cells selected for resistance to NSC73306 revealed loss of P-gp expression and consequent loss of the MDR phenotype. Although hypersensitivity to NSC73306 required functional expression of P-gp, biochemical assays revealed no direct interaction between NSC73306 and P-gp. This article shows that NSC73306 kills cells with intrinsic or acquired P-gp-induced MDR and indirectly acts to eliminate resistance to MDR1 substrates.

Cisplatin resistance has been associated with altered K+ fluxes. Here, we focused our investigations on the detection of K+ channels in a series of cisplatin-resistant (CP-r) cells with increasing resistance and on the functional relationship of these K+ channels to resistance. Microarray analysis and confocal microscopy indicated that there was overexpression of the ether-a-gogo gene (HERG) and the inwardly rectifying potassium channel gene (TWIK) in a human epidermal KB and human liver BEL-7404 carcinoma cell line series selected for cisplatin resistance. With increased resistance, the plasma membrane potential, but not the mitochondrial membrane potential, also increases in these two series. For these reasons, we conducted cell proliferation studies in the presence of either antibodies directed against the detected K+ channels, omeprazole (a H+ pump inhibitor) or a specific inhibitor of the HERG channel (WAY-123398-A-5). The antibodies and omeprazole influenced cell growth only very slightly. The specific K+ channel blocker did not alter cisplatin resistance. We also observed that manipulation of K+ fluxes with antibodies and the H+ pump with omeprazole resulted in opposite effects on cisplatin resistance in these two cell lines. We conclude that K+ and H+ homeostasis are not critical factors in cisplatin resistance since they affect cisplatin resistance differently in KB and BEL-7404 cells.

Discovery of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) transporter able to transport many anticancer drugs, was a clinically relevant breakthrough in multidrug resistance research. Although the overexpression of ABC transporters such as P-glycoprotein/ABCB1, MRP1/ABCC1, and MXR/ABCG2 seems to be a major cause of failure in the treatment of cancer, acquired resistance to multiple anticancer drugs may also be multifactorial, involving alteration of detoxification processes, apoptosis, DNA repair, drug uptake, and overexpression of other ABC transporters. As a tool for the study of such phenomena, we designed and created a microarray platform, the ABC-ToxChip, to evaluate relative levels of transcriptional activation among genes involved in the various mechanisms of resistance. In the ABC-ToxChip, a comprehensive set of genes important in toxicological responses (represented by 2200 cDNA probes) is complemented with probes specifically matching ABC transporters as well as oligonucleotides representing 18,000 unique human genes. By comparing the transcriptional profiles of KB-3-1 and DU-145 parental cells with resistant derivatives selected in colchicine (KB-8-5), and 9-nitro-camptothecin (RCO.1), respectively, we demonstrate that ABC transporters (ABCB1/MDR1 and ABCC2/MRP2, respectively) show dramatic overexpression, whereas the glutathione S-transferase gene GST-Pi shows the strongest decrease in expression among the 20,000 genes studied. The results were confirmed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The custom-designed ABC-Tox microarray presented here will be helpful to elucidate mechanisms leading to anticancer drug resistance.

The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance.

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DMA. In both drug-sensitive 3T3 cells and drug-resistant 3T3 cells (NIH-MDR-6185, created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells) initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells, and accumulated in G(2)/M. The 2 to 3 h lag period represents the time taken for fully establishing the G(2)/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G] and S, and did not reach G(2)/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G(2)/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).

The FDA approved HIV-1 protease inhibitors, ritonavir, saquinavir, and indinavir, are very effective in inhibiting HIV-1 replication, but their long-term efficacy is unknown. Since in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether these protease inhibitors are recognized by the MDR1 multidrug transporter (P-glycoprotein, or P-gp), thereby reducing their intracellular accumulation. In vitro studies in isolated membrane preparations from insect cells infected with MDR1-expressing recombinant baculovirus showed that these inhibitors significantly stimulated P-gp-specific ATPase activity and that this stimulation was inhibited by SDZ PSC 833, a potent inhibitor of P-gp. Furthermore, photoaffinity labeling of P-gp with the substrate analogue [125I]iodoarylazidoprazosin (IAAP) was inhibited by all three inhibitors. Cell-based approaches to evaluate the ability of these protease inhibitors to compete for transport of known P-gp substrates showed that all three HIV-1 protease inhibitors were capable of inhibiting the transport of some of the known P-gp substrates but their effects were generally weaker than other documented P-gp modulators such as verapamil or cyclosporin A. Inhibition of HIV-1 replication by all three protease inhibitors was reduced but could be restored by MDR1 inhibitors in cells expressing MDR1. These results indicate that the HIV-1 protease inhibitors are substrates of the human multidrug transporter, suggesting that cells in patients that express the MDR1 transporter will be relatively resistant to the anti-viral effects of the HIV-1 protease inhibitors, and that absorption, excretion, and distribution of these inhibitors in the body may be affected by the multidrug transporter.

Human Pgp from the vinblastine-resistant cell line, KB-V1, can be purified by sequential conventional chromatography on DEAE-sepharose CL-6B resin followed by a wheat germ agglutinin column. By including glycerol (osmolyte protectant) and lipid during the solubilization and chromatography procedures most of the biological activity of Pgp can be retained. The activity of Pgp in the detergent extract or in the concentrated column fractions is stable for at least 8-10 months when stored at -80 degrees. However, repeated cycles of freezing and thawing of fractions result in considerable loss of activity. We have purified Pgp from KB-C1 (a subclone of KB 3-1 that is resistant to 1 microgram/ml colchicine) by following the same protocol. When this method was used for purification of Pgp from MDR1-transfected NIH 3T3 transfectants (N3-V2400, grown in the presence of 2.4 micrograms/ml vinblastine), the protein was eluted with 0.1 M NaCl from the DEAE-Sepharose CL-6B column as usual. However, during WGA lectin chromatography, the protein was eluted with a lower concentration of sugar (0.1 M instead of 0.25 M NAG). This altered elution pattern appears to be due to a difference in the glycosylation of human Pgp in mouse NIH 3T3 cells. This is consistent with the observation that human Pgp expressed in NIH 3T3 cells migrates faster compared to the protein from KB-V1 cells on 8-10% acrylamide gel. Similarly, other workers have purified Chinese hamster Pgp either by a single-step chromatography on Reactive Red 120 agarose or by a combination of anion exchange and immunoaffinity chromatography (see the article by Senior et al. for the purification and properties of ATPase activity of Chinese hamster Pgp). The high level of drug-stimulated ATP hydrolysis by Pgp (Table I), like other ion-transporting ATPases, indicates that this is a high-capacity pump that can function as an effective multidrug transporter. This is further supported by the qualitative demonstration of ATP-dependent vinblastine transport in proteoliposomes reconstituted with pure Pgp (see Fig. 2). Thus, these experiments provide strong evidence that purified Pgp retains its activity and that it functions as an ATP-dependent drug transporter.

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

In this work, we demonstrate a protective effect conferred by the human multidrug resistance gene (MDR1) to populations of the murine hematopoietic system against the toxic effects of bisantrene, a novel intercalating cytotoxic agent under investigation as an anticancer agent. In vitro, MDR1-expressing cell lines are highly cross-resistant to bisantrene, and low levels of P-glycoprotein (the MDR1 gene product cell surface protein) confer resistance to the drug. MDR1-positive mice were generated after transplantation of bone marrow cells (BMC) transduced in vitro with a MDR1 retrovirus. Control mice were transplanted with BMC transduced with the neomycin resistance gene. Administration of a single i.v. dose of 50 mg/kg of bisantrene resulted in a decrease of the total WBC count of approximately 40%. In contrast, a decrease of the total WBC count of only 17% was observed in mice transplanted with MDR1-transduced BMC. Immunofluorescence studies with cell lineage-specific monoclonal antibodies showed that bisantrene was specifically toxic for B lymphocytes and macrophages. Double-staining with MRK16 (a monoclonal antibody specific for P-glycoprotein) demonstrated that a single dose of bisantrene increased the relative number of MDR1-transduced positive B cells, macrophages, and (to some extent) granulocytes when compared to the number found in MDR1-untreated mice or the bisantrene-treated neomycin-transduced control mice. These results provide in vivo evidence that bisantrene is a hematotoxic drug capable of selecting for MDR1-transduced hematopoietic cells. Bisantrene might be useful for gene therapy as an in vivo selective agent for cells transduced with MDR1 vectors that also coexpress therapeutic genes of interest.

Multidrug resistance (MDR) may be associated with the expression of the MDR1 gene which encodes the 170-kDa cell surface P-glycoprotein (PGP) acting as an energy-dependent multidrug efflux pump. This pump can be inhibited by a variety of drugs including cyclosporin A, quinidine, and verapamil. Substrate specificity of the MDR1 gene product can be altered by a point mutation at amino acid residue 185 in which valine is substituted for glycine, but the effect of this mutation on inhibition of PGP is unknown. Multidrug-resistant NIH3T3 cells transfected with the MDR1 retroviral vector pHaMDR-1/A (G185) or pHaMDR1/A (V185) expressing comparable levels of PGP were compared for patterns of drug resistance and inhibition of drug resistance by MDR reversing agents. The NIH-MDR-G185 transfectants were somewhat preferentially resistant to daunorubicin, taxol, and vinblastine. The mutant (V185) conferred increased resistance to colchicine. This MDR phenotype in both NIH-MDR-G185- and NIH-MDR-V185-transfected NIH3T3 cells was overcome by the addition of cyclosporin A, quinidine, or verapamil. Verapamil was the most potent of the three agents affecting wild-type PGP. However, specific inhibitors showed different potency with wild-type or mutant transporters, depending on the cytotoxic drug whose resistance was being reversed. For example, cyclosporin A at a concentration of 1 microgram/ml, was a powerful reverser of taxol and colchicine resistance for the mutant drug transporter, but was much less effective for the wild-type transporter. In contrast, verapamil reversed resistance to vinblastine more efficiently for the wild-type transporter than for the mutant transporter. These results suggest that the sensitivity of a multidrug transporter to a reversing agent will depend on the reversing agent, the cytotoxic drug, and the presence or absence of mutations which alter substrate specificity.

We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 [Lelong et al. (1992) FEBS Lett. 304, 256-260]. Like [gamma-32P]ATP, [gamma-32P]GTP was also able to phosphorylate Pgp in vitro. Unlabeled GTP enhanced the phosphorylation of the transporter by [gamma-32P]ATP, whereas unlabeled ATP inhibited incorporation of label. While phosphorylation by [gamma-32P]ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+. Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation. The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter. Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides. These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.

Two general approaches to the gene therapy of cancer have been proposed: (1) strategies that use exogenous genes to modify cancer cells so that they are less malignant or more susceptible to host defenses or to killing by exogenous agents; and (2) approaches that modify host cells so that they are more effective in eliminating cancer cells or more resistant to agents that are used to treat cancer. In both cases, the development of vectors that encode in vivo selectable phenotypes, such as drug resistance, would be extremely valuable because of the inherent inefficiency of gene transfer and the potential of such vectors to protect normal tissues against toxic agents. To allow the selection of cells in vivo that have been transduced with vectors for gene therapy, we have utilized the human multidrug resistance (MDR1) gene. The product of this gene is a 170,000-dalton glycoprotein known as P-glycoprotein, which acts as an energy-dependent efflux pump for a great many cytotoxic anticancer drugs, including doxorubicin, daunorubicin, etoposide, teniposide, actinomycin D, and taxol. Vectors encoding an MDR1 cDNA are able to transduce many cell types, including bone marrow cells, with high efficiency to allow selection of drug resistance in vitro and in vivo in mouse models. Thus, it should be possible to protect the bone marrow of patients undergoing intensive chemotherapy by transduction of their bone marrow with MDR1 vectors. Furthermore, the ability to select for the presence of the MDR1 cDNA in vivo means that it can be used to introduce otherwise nonselectable genes into the bone marrow for therapy of cancer and other diseases.

The antifungal agent ketoconazole was found to overcome resistance to vinblastine and doxorubicin in multidrug resistant KB-V1 cells in vitro. These cells are several hundred-fold more resistant than the parental cell line KB-3-1. Ketoconazole had little or no effect on the parental KB-3-1 cells. The concentrations used to overcome drug resistance in vitro have already been safely used in vivo for treatment of fungal infections and in the monotherapy of hormone independent prostate carcinomas to block adrenal androgen production. Because of a possible beneficial effect of a combination of ketoconazole and a chemotherapeutic drug in multidrug resistant cancers, we examined a panel of 11 prostate carcinoma tissues for the expression of the MDR1 gene by an RNA-PCR assay. MDR1 expression was detectable, albeit at low levels, in 8 of the 11 tumors, suggesting a possible role of this gene in the drug resistance of prostate carcinomas. Our data suggest that ketoconazole might be useful in overcoming multidrug resistance in concentrations that are achievable in humans.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.

Levamisole, originally developed as an anthelmintic, is used in the treatment of many disorders. Severe side-effects have been noted by some investigators, and a case is presented herein of levamisole-induced agranulocytosis.

The accumulation of daunomycin in drug-sensitive and multidrug-resistant human KB cells was examined using light microscopy to detect the inherent fluorescence of daunomycin. Intracellular accumulation of fluorescent drug occurred rapidly in parental KB cells and was markedly reduced in several multidrug-resistant mutants. The addition of verapamil, which reverses multidrug resistance, resulted in increased accumulation of daunomycin in resistant cells. In living cells, most of the daunomycin was found in the nucleus, but significant amounts were detected associated with the plasma membrane, in the cytoplasm, in organelles of the Golgi region, and in lysosomes. The nuclear fluorescence was measured using a photometer system, and the loss of daunomycin from the cells was determined under various conditions. When sensitive cells were allowed to accumulate daunomycin for 5 min at 37 degrees C and then placed in medium without the drug, daunomycin remained inside the nuclei for longer than 1 day. When resistant cells were loaded in the presence of verapamil and the verapamil was removed, the resistant cells lost daunomycin with a half-time of about 1 min. The continuous presence of verapamil markedly inhibited the loss of daunomycin from the cells. Similar results were obtained in separate experiments using [3H]daunomycin. Vinblastine, vincristine, and quinidine were also effective in stimulating daunomycin accumulation in multidrug-resistant cells and in preventing the loss of daunomycin from these resistant cells. This effect required half-maximal concentrations of 1-2 microM for verapamil, vinblastine, and quinidine. Ouabain, lanthanum, colchicine, amiloride, probenecid, and 1-beta-D-arabinofuranosylcytosine had no effect on this process. Quinine was effective at 10 microM and nifedipine was effective at 50 microM. Depletion of cellular adenosine triphosphate levels by preincubation of cells with azide and 2-deoxyglucose partially inhibited daunomycin loss from resistant cells. These single-cell measurements indicate that diminished daunomycin accumulation in multidrug-resistant cells results from accelerated energy-dependent efflux across the plasma membrane, and this efflux is inhibited by verapamil, quinidine, vincristine, and vinblastine.

Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor (EGF) and Pseudomonas exotoxin (PE) were selected. EGF-PE and the drug verapamil, which enhanced EGF-PE cytotoxicity, were used in the selection process. These mutants also showed some cross-resistance to PE. All of the EGF-PE resistant variants displayed lower levels of 125I-EGF binding, 20-50% of parental KB levels, without altered affinity for EGF and grew at a slower rate than the parental cell line KB-3-1. These results indicate that EGF-PE resistant KB cells have a complex phenotype which includes a reduction in the number of EGF receptors and reduced sensitivity to unconjugated PE. Resistance to toxin-conjugates, although pleiotropic, is specific and does not lead to resistance to multiple other anticancer drugs, nor are independently selected multidrug resistant KB lines resistant to PE. These results argue that protocols for cancer treatment could effectively use specifically designed cytotoxic toxin conjugates as an adjunct to conventional chemotherapy.