Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Advanced Autologous Lower Dermal Sling Technique for Immediate Breast Reconstruction Surgery in Small and Non-ptotic Breasts.

Breast reconstruction with an autologous lower dermal sling (ALDS) is an established one-stage procedure in patients with moderate to large ptotic breasts. However, this technique is difficult to perform in small and non/minimally ptotic breasts. We describe our experiences from a single institution about a novel Advanced Autologous Lower Dermal Sling (A-ALDS) technique for reconstruction in small breasts. We performed one-stage nipple/skin sparing mastectomies in 61 patients with immediate reconstruction either by Conventional Immediate Breast Reconstruction Surgery or A-ALDS technique. Mean age of study patients was 46.9 years. We observed significantly better cosmetic score and lower immediate complication rate vis-a-vis skin necrosis, implant loss with the A-ALDS technique (i.e., nil versus 3 in Conventional Immediate Breast Reconstruction Surgery (IBRS)). Forty patients completed 12-month follow-up. The PROMs - Patient Reported Outcome Measures (Breast-Q) revealed good to excellent scores for satisfaction with breast, cosmetic outcome, and psychosocial well-being in patients operated with both these techniques. However, sexual well-being was significantly better in the A-ALDS group. The A-ALDS is a novel, cost-effective, and safe technique for immediate one-stage implant-based reconstruction for small breasts. It provides a dermal barrier flap and hence, ensures less complications, excellent cosmetic results, and patient satisfaction. Supplementary Information: The online version contains supplementary material available at 10.1007/s13193-022-01524-8.

Cancer Patient Management during COVID-19 Pandemic: An Audit of a Single-Surgeon Unit in a COVID-Hotspot.

The report evaluates the effect of coronavirus disease (COVID-19) pandemic on breast cancer treatment and management at a single-surgeon cancer care unit in one of the hotspots of COVID-19 in India. In response to the pandemic, the adjustments were made in the clinical practice to accommodate social distancing. Patient consultations were done over phone call or in-clinic visit with prior appointment to reduce the risk of exposure to COVID-19. Total number of patients that were treated at the clinic and the essential surgeries performed during the pandemic phases are summarized in the report. The methodology adopted here for care and management of the cancer patients can serve as a guiding principle for cancer care units in the country.

The 2nd Conference and Workshop of The Cancer Genome Atlas (TCGA) in India: Towards Team Science for Multi-omics Cancer Research in South Asia.

The Cancer Genome Atlas (TCGA) is a landmark cancer genomics program that molecularly characterized over 20,000 primary cancer and matched normal samples spanning 33 cancer types. On similar lines, the establishment of an 'Indian Cancer Genomics Atlas (ICGA)' has been initiated in 2019-2020 by a consortium of key stakeholders in India led by Council for Scientific and Industrial Research (CSIR), Government of India and several reputed governmental agencies, cancer hospitals, academic institutions, and private sector partners. In parallel, Bangladesh Medical Research Council (BMRC) has announced the launch of 'Bangladesh Cancer Genome Atlas (BCGA) project with support from the ICGA teams. Teams from United States - National Cancer Institute (NCI) office of TCGA and Centre for Global Oncology, Institute of Cancer Research, London, United Kingdom are interested in extending their collaborations to these large-scale initiatives by acting as knowledge partners. With this background, an online version of the 2nd TCGA conference and workshop in India was organised with the theme of 'Towards Team Science for Multi-omics Studies in South Asia' on December 3-5, 2020. Over 1,500 delegates comprising of onco-clinicians, basic researchers, bioinformaticians, geneticists, translational researchers, big-data and machine-learning scientists, bioethicists and regulatory experts from across the globe attended the event. The conference agenda focused on the vision, design and plans of the ICGA project with regards to common standard operating protocols (SOPs), operations, logistics, bioethics, policy and governance models. More importantly, conference sessions were planned around the central theme of building a culture of team science for undertaking mega-cancer research projects in India and neighbouring countries. Experts from the globe deliberated on the latest technical aspects of data/biospecimen/multi-omics studies and applications of Precision Oncology in clinical cancer management.

Breast cancer biobank from a single institutional cohort in an urban setting in india: Tumor characteristics and survival outcomes.

BACKGROUND: A breast cancer biobank with retrospectively collected patient data and FFPE tissue samples was established in 2018 at Prashanti Cancer Care Mission, Pune, India. It runs a cancer care clinic with support from a single surgeon's breast cancer practice. The clinical data and tissue sample collection is undertaken with appropriate patient consent following ethical approval and guidelines. METHODS: The biobank holds clinical history, diagnostic reports, treatment and follow-up information along with FFPE tumor tissue specimens, adjacent normal and, in few cases, contralateral normal breast tissue. Detailed family history and germline mutational profiles of eligible and consenting patients and their relatives are also deposited in the biobank. RESULTS: Here, we report the first audit of the biobank. A total number of 994 patients with breast disease have deposited consented clinical records in the biobank. The majority of the records (80%, n = 799) are of patients with infiltrating ductal carcinoma (IDC). Of 799 IDC patients, 434 (55%) have deposited tumor tissue in the biobank with consent. In addition, germline mutation profiles of 84 patients and their family members are deposited. Follow-up information is available for 85% of the 434 IDC patients with an average follow-up of 3 years. CONCLUSION: The biobank has aided the initiation of translational research at our center in collaboration with eminent institutes like IISER Pune and SJRI Bangalore to evaluate profiles of breast cancer in an Indian cohort. The biobank will be a valuable resource to the breast cancer research community, especially to understand South Asian profiles of breast cancer.

Withania somnifera (L.) Dunal: Opportunity for Clinical Repurposing in COVID-19 Management.

As the COVID-19 pandemic is progressing, the therapeutic gaps in conventional management have highlighted the need for the integration of traditional knowledge systems with modern medicine. Ayurvedic medicines, especially Ashwagandha (Withania somnifera (L.) Dunal, WS), may be beneficial in the management of COVID-19. WS is a widely prescribed Ayurvedic botanical known as an immunomodulatory, antiviral, anti-inflammatory, and adaptogenic agent. The chemical profile and pharmacological activities of WS have been extensively reported. Several clinical studies have reported its safety for use in humans. This review presents a research synthesis of in silico, in vitro, in vivo, and clinical studies on Withania somnifera (L.) Dunal (WS) and discusses its potential for prophylaxis and management of COVID-19. We have collated the data from studies on WS that focused on viral infections (HIV, HSV, H1N1 influenza, etc.) and noncommunicable diseases (hypertension, diabetes, cancer, etc.). The experimental literature indicates that WS has the potential for 1) maintaining immune homeostasis, 2) regulating inflammation, 3) suppressing pro-inflammatory cytokines, 4) organ protection (nervous system, heart, lung, liver, and kidney), and 5) anti-stress, antihypertensive, and antidiabetic activities. Using these trends, the review presents a triangulation of Ayurveda wisdom, pharmacological properties, and COVID-19 pathophysiology ranging from viral entry to end-stage acute respiratory distress syndrome (ARDS). The review proposes WS as a potential therapeutic adjuvant for various stages of COVID-19 management. WS may also have beneficial effects on comorbidities associated with the COVID-19. However, systematic studies are needed to realize the potential of WS for improving clinical outcome of patients with COVID-19.

NGS-based profiling of key cancer genes in Indian triple-negative breast cancer patients reinforces molecular heterogeneity of the disease.

Triple-negative breast cancers (TNBC) are one of the most aggressive forms of breast cancers. With poor patient outcomes, it presents a great burden on the healthcare systems. There have been some efforts to explore the genomic changes that occur in TNBCs. However, there is not enough data on Indian TNBCs. We sought to understand the mutational landscape of key cancer-associated genes in Indian TNBC patients using TruSeq Cancer Amplicon Panel. We sequenced 51 TNBC patient samples and found great heterogeneity amongst samples with respect to the genomic variants. Several previously reported including alterations in PI3K-AKT pathway genes were also identified. Likewise, we identified several novel high-frequency variants, for example, GNAQ F341S (17%), the functional role of which remains unclear. Our study lays the foundation of larger efforts needed to understand the genomic landscape of Indian TNBCs which can aid in classification and better therapeutic management of patients.

Breast conservation surgery & oncoplasty in India - Current scenario.

Breast cancer incidence is on the rise in India as in rest of the world. While the advances in overall cancer care are at par, the surgical interventions have not been changing at the same pace in India, as in the rest of the developed world. Partly, this is due to the relatively more advanced state of cancer at detection and partly due to lack of awareness resulting in apprehension and slow acceptance of de-escalation of surgical interventions by the treating surgeons, and the beneficiaries, the patients. The article looks at the current scenario, available evidence on the practices and pitfalls with possible solutions for advancing surgical care of breast cancer in India.

A blood transcriptome-based analysis of disease progression, immune regulation, and symptoms in coronavirus-infected patients.

COVID-19 patients show heterogeneity in clinical presentation and outcomes that makes pandemic control and strategy difficult; optimizing management requires a systems biology approach of understanding the disease. Here we sought to potentially understand and infer complex disease progression, immune regulation, and symptoms in patients infected with coronaviruses (35 SARS-CoV and 3 SARS-CoV-2 patients and 57 samples) at two different disease progression stages. Further, we compared coronavirus data with healthy individuals (n = 16) and patients with other infections (n = 144; all publicly available data). We applied inferential statistics (the COVID-engine platform) to RNA profiles (from limited number of samples) derived from peripheral blood mononuclear cells (PBMCs). Compared to healthy individuals, a subset of integrated blood-based gene profiles (signatures) distinguished acute-like (mimicking coronavirus-infected patients with prolonged hospitalization) from recovering-like patients. These signatures also hierarchically represented multiple (at the system level) parameters associated with PBMC including dysregulated cytokines, genes, pathways, networks of pathways/concepts, immune status, and cell types. Proof-of-principle observations included PBMC-based increases in cytokine storm-associated IL6, enhanced innate immunity (macrophages and neutrophils), and lower adaptive T and B cell immunity in patients with acute-like disease compared to those with recovery-like disease. Patients in the recovery-like stage showed significantly enhanced TNF, IFN-γ, anti-viral, HLA-DQA1, and HLA-F gene expression and cytolytic activity, and reduced pro-viral gene expression compared to those in the acute-like stage in PBMC. Besides, our analysis revealed overlapping genes associated with potential comorbidities (associated diabetes) and disease-like conditions (associated with thromboembolism, pneumonia, lung disease, and septicemia). Overall, our COVID-engine inferential statistics platform and study involving PBMC-based RNA profiling may help understand complex and variable system-wide responses displayed by coronavirus-infected patients with further validation.

Withania somnifera (L.) Dunal: A potential therapeutic adjuvant in cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal (WS) is one of the moststudied Rasayana botanicals used in Ayurveda practice for its immunomodulatory, anti-aging, adaptogenic, and rejuvenating effects. The botanical is being used for various clinical indications, including cancer. Several studies exploring molecular mechanisms of WS suggest its possible role in improving clinical outcomes in cancer management. Therefore, research on WS may offer new insights in rational development of therapeutic adjuvants for cancer. AIM OF THIS REVIEW: The review aims at providing a detailed analysis of in silico, in vitro, in vivo, and clinical studies related to WS and cancer. It suggests possible role of WS in regulating molecular mechanisms associated with carcinogenesis. The review discusses potential of WS in cancer management in terms of cancer prevention, anti-cancer activity, and enhancing efficacy of cancer therapeutics. MATERIAL AND METHODS: The present narrative review offers a critical analysis of published literature on WS studies in cancer. The reported studies were analysed in the context of pathophysiology of cancer, commonly referred as 'cancer hallmarks'. The review attempts to bridge Ayurveda knowledge with biological insights into molecular mechanisms of cancer. RESULTS: Critical analysisof the published literature suggests an anti-cancer potential of WS with a key role in cancer prevention. The possible mechanisms for these effects are associated with the modulation of apoptotic, proliferative, and metastatic markers in cancer. WS can attenuate inflammatory responses and enzymes involved in invasion and metastatic progression of cancer.The properties of WS are likely to be mediated through withanolides, which may activate tumor suppressor proteins to restrict proliferation of cancer cells. Withanolides also regulate the genomic instability, and energy metabolism of cancer cells. The reported studies indicate the need for deeper understanding of molecular mechanisms of WS in inhibiting angiogenesis and promoting immunosurveillance. Additionally, WS can augment efficacy and safety of cancer therapeutics. CONCLUSION: The experimentally-supported evidence of immunomodulatory, anti-cancer, adaptogenic, and regenerative attributes of WS suggest its therapeutic adjuvant potential in cancer management. The adjuvant properties of withanolides can modulate multidrug resistance and reverse chemotherapy-induced myelosuppression. These mechanisms need to be further explored in systematically designed translational and clinical studies that will pave the way for integration of WS as a therapeutic adjuvant in cancer management.

Primary Angiosarcoma Of the Breast: A Case Report.

Primary angiosarcoma of the breast is a rare (0.04% of all malignant breast tumors) and potentially life-threatening disease. Given its variable and non-specific clinical, radiological and pathological presentation, accurate diagnosis is a challenge. Primary angiosarcoma of the breast predominantly occurs in younger patients and it is often overlooked and misdiagnosed at radiology and pathology. To ensure that this aggressive malignancy is not overlooked, radiologists need to be aware of the fact that such tumors may present with non-specific imaging features. We report a case of a 32-year-old female with primary angiosarcoma of the breast presenting with non-specific imaging features. It was initially interpreted as a capillary cavernous hemangioma at histopathology following an ultrasound-guided biopsy. This eventually turned out to be angiosarcoma after a second histopathology opinion was sought in light of the radiology-pathology discordance.

Extreme Oncoplastic Surgery for Multifocal/Multicentric and Locally Advanced Breast Cancer.

INTRODUCTION: Breast conserving surgery (BCS) followed by radiation therapy (RT) has become the preferred alternative to mastectomy for patients with early stage breast cancer (BC). Randomized trials have confirmed equivalent locoregional control and overall survival for BCS and mastectomy. Extreme Oncoplasty (EO) extends the indications of BCS for patients who would otherwise require mastectomy, ensuring better aesthetic outcomes and oncological safety. METHODS: BC patients with multifocal/multicentric (MF/MC) tumors, extensive DCIS, or large tumor >50mm underwent EO at our breast unit. Therapeutic reduction mammaplasty (TRM) with wise pattern preoperative markings and dual pedicle technique involving parenchymal rearrangement was used for oncoplastic reconstructions in majority of the cases followed by RT. Patient reported outcome measures (PROMs) were assessed using the validated Breast-Q questionnaire. RESULTS: Of the 39 patients in the study, 36 had unilateral and 3 had bilateral BC. Mean age was 47.2 years. Median tumor size was 75mm. 17 (43.6%) patients received NACT; none achieved a complete clinical response. 28 (71.8%) patients were administered to adjuvant chemotherapy. 33(84.6%) patients received RT to the breast with a median dose of 50Gy in 28 fractions and a boost dose of 10Gy in 5 fractions to the tumor bed. No major complications or local recurrences were observed. Excellent Breast-Q scores were observed in patients undergoing EO after 12 months of follow-up. CONCLUSION: EO followed by RT results in acceptable local-regional control, low rate of complications, and high patient satisfaction. In selected patients, EO could provide a safe alternative for breast conservation surgery instead of mastectomy.

Management of chronic generalized periodontitis using diode laser.

The aim of this study was to evaluate the effect of a diode laser with nonsurgical periodontal therapy on chronic periodontitis. The patient, a 37-year-old female, with chronic periodontitis reported to the private dental clinic. Her health history indicated that she had good general health. The periodontal examination included a gingival index and complete periodontal probing depth with William's graduated probe. She was treated with 940 nm diode laser and scaling and root planning. Assessment was done after 6 months following laser therapy; the probing depths improved; gain in clinical attachment levels; no inflammation; the tissue tone was good, showing increased stippling.

Patient-Reported Outcomes Following Breast Reconstruction Surgery and Therapeutic Mammoplasty: Prospective Evaluation 1 Year Post-Surgery with BREAST-Q Questionnaire.

Breast Cancer (BC) treatment leads to mutilation and destruction of breast shape with negative effects on body image and self-esteem.One of the main goals of reconstructive and oncoplastic breast surgery is to satisfy patients and improve their quality of life (QoL).Therefore, it is important to assess the patient experience post-surgery by means of patient-reported outcome measures (PROMs) that focus on the patient's perception of the surgery and surgical care, as well as psychosocial well-being and physical functioning. The objective of the current study was to identify predictors of patient satisfaction such as breast appearance including implant type in a selective sample of women who underwent breast reconstruction surgery using implants. Participants in this prospective study were women, (age 26-75 years) that were newly diagnosed with breast carcinoma. All consecutive patients who underwent breast reconstruction between January 2013 and October 2014 were asked to complete the BREAST-Q questionnaire 1 year after surgery. 120 patients underwent unilateral breast reconstruction using implant. While 38 patients underwent reconstruction with opposite breast reduction symmertization, 27 patients underwent therapeutic mammoplasty. The response rate for BREAST-Q questionnaire completion was 98 % with 147 out of 150 study participants completed the questionnaire. From the data collected from 147 patients, the responses could be distributed into 4 distinct groups based on the reconstruction outcomes namely "very much satisfied" (93 %) or "definitely and mostly satisfied" (94 %) or "satisfied" with the outcome (88 %) or "definitely agree on having reconstruction rather than the alternative of having no breast "(91 %).The results showed significant improvement in all four areas that were evaluated after surgery namely satisfaction with the appearance of the breasts, psychosocial, sexual and physical well-being. While the reconstruction surgery had an overall positive impact on quality of life it was observed that in patients that did not undergo breast reconstruction psychological issues related to sexuality were observed. Many newly diagnosed BC patients do not opt for these new surgical procedures due to psychological trauma or lack of information. Based on these observations, we propose that Breast Cancer Management protocols should also include additional counselling support for the newly diagnosed BC patients on the option of breast reconstruction along with conventional medical treatment counselling by the Oncosurgeon.

The influence of tray space and repeat pours on the accuracy of monophasic polyvinylsiloxane impression.

AIM: While literature demonstrates that the optimum accuracy is obtained with the custom trays, the use of stock trays for elastomeric impressions appears to be popular in general practice. The aim of the study was to evaluate the effect of thickness of impression material on the dimensional accuracy of impressions made from monophasic polyvinylsiloxane impression material. This study also studies the dimensional stability of the impressions poured at different time intervals. MATERIALS AND METHODS: A metal model simulating two abutment teeth was fabricated along with reference lines inscribed on them. Custom impression trays were fabricated with spacer thickness of 2, 4 and 6 mm. impressions were made using monophasic polyvinyl siloxane impression material. The impressions were poured and stone models were obtained. The dimensional accuracy of the impressions were determined indirectly by measuring the dimensional changes of the recovered stone models. The dimensional stability was also evaluated by pouring the impressions at time intervals of 1 hour, 24 hours and 7 days. The obtained data was statistically analyzed. RESULTS: The results of the study indicated that the impressions made from 2 and 4 mm tray space produced more accurate stone models when compared to 6 mm tray space. There was no significant deviation in the mesiodistal and buccolingual dimensions when impressions were made using impression trays with 2, 4 and 6 mm tray spacers. There was a significant decrease in interabutment distance (p = 0.001) and height of the abutment (p = 0.024) when impressions were made using impression trays with a tray space of 6 mm. There were no significant differences found among the stone models obtained from 1 hour, 24 hours and 1 week pour times. CONCLUSION: The mesiodistal and buccolingual dimensions of the abutments were not affected by the variations in thickness of impression material. There was a decrease in the height of the abutment which was clinically not significant. As the thickness of the impression material increased, the interabutment space decreased, which is a factor of concern when impressions are made for fixed partial dentures. CLINICAL SIGNIFICANCE: When monophasic polyvinylsiloxane is used as the impression material, stock trays can be used for making impressions for individual castings.

The role of pregnane X receptor in 2-acetylaminofluorene-mediated induction of drug transport and -metabolizing enzymes in mice.

Activation of the pregnane X receptor (PXR) mediates the induction of several drug transporters and -metabolizing enzymes. In vitro studies have reported that several of these genes are induced after exposure to the hepatocarcinogen, 2-acetylaminofluorene (2-AAF). Thus, we hypothesized that PXR may play a role in the in vivo induction of gene expression by 2-AAF. We examined the expression of the drug-metabolizing enzymes CYP1A2 and CYP3A11 and the drug transporters breast cancer resistance protein (BCRP), MRP2, and OATP2. Wild-type (PXR+/+) and PXR-null (PXR-/-) C57BL/6 mice were injected daily for 7 days with 150 or 300 mg/kg 2-AAF suspended in corn oil (i.p.), whereas the control group received corn oil vehicle. Levels of mRNA isolated from liver were measured by reverse transcription-polymerase chain reaction and normalized to beta-actin. Treatment of PXR+/+ mice resulted in a dose-dependent 2- to 4-fold induction (p<0.001) of MRP2, OATP2, BCRP, CYP3A11, and CYP1A2, but no induction was observed in PXR-/- mice. Induction of PXR mRNA was observed in the 2-AAF-treated PXR+/+ mice. Furthermore, a dose-dependent increase in CYP3A4 promoter construct activity was observed in HepG2 cells cotransfected with human or rat PXR, indicating that 2-AAF does indeed activate PXR. These results suggest that PXR is responsible for 2-AAF-mediated induction of drug efflux transporters and biotransformation enzymes in the liver. Moreover, novel findings demonstrate that PXR plays a role in regulation of the drug efflux transporter, BCRP, in mice.

Beyond CAR and PXR.

It is becoming increasingly evident that constitutive, induced, and regulated expression of genes important to the drug disposition process such as drug transporters, phase I and II metabolic enzymes are largely under the transcriptional control of certain nuclear receptor (NR) family members. In the past decade, important new insights regarding the role and relevance of ligand-activated nuclear receptors such as such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in terms of their activation by endogenous biochemicals, natural products, as well as synthetic compounds have led to a much better understanding of the xenobiotic-mediated induction process and the clinical relevance of such NRs to drug therapy in general. However, in addition to CAR and PXR, many orphan and adopted orphan NRs have recently been identified as key regulators of drug disposition genes. Indeed, nuclear receptors including farnesoid X receptor, peroxisome proliferator-activated receptor, and hepatocyte nuclear factors (1alpha, 3 and 4alpha) exhibit overlapping ligand specificities and regulate multiple gene targets, resulting in tissue- and organ-specific expression of drug disposition genes. In this review, the biology, pathophysiology, and the potential clinical relevance of such NRs to drug disposition and response are discussed.

Nitric oxide mediates increased P-glycoprotein activity in interferon-{gamma}-stimulated human intestinal cells.

Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-gamma and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-gamma reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-gamma stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-gamma-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-gamma and SNAP enhanced DNA binding of NF-kappaB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-kappaB binding. These results suggest that NO mediates IFN-gamma-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-kappaB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.