Potential Impact of PI3K-AKT Signaling Pathway Genes, KLF-14, MDM4, miRNAs 27a, miRNA-196a Genetic Alterations in the Predisposition and Progression of Breast Cancer Patients.

Genome-wide association studies have reported link between SNPs and risk of breast cancer. This study investigated the association of the selected gene variants by predicting them as possible target genes. Molecular technique advances with the availability of whole-exome sequencing (WES), now offer opportunities for simultaneous investigations of many genes. The experimental protocol for PI3K, AKT-1, KLF-14, MDM4, miRNAs 27a, and miR-196a genotyping was done by ARMS-PCR and sanger sequencing. The novel and known gene variants were studied by Whole-exome sequencing using Illumina NovaSeq 6000 platform. This case control study reports significant association between BC patients, healthy controls with the polymorphic variants of PI3K C > T, AKT-1 G > A KLF 14 C > T, MDM4 A > G, miR-27a A > G, miR-196a-2 C > T genes (p < 0.05). MDM4 A > G genotypes were strongly associated with BC predisposition with OR 2.08 & 2.15, p < 0.05) in codominant and dominant models respectively. MDM4 A allele show the same effective (OR1.76, p < 0.05) whereas it remains protective in recessive model for BC risk. AKT1G > A genotypes were strongly associated with the BC susceptibility in all genetic models whereas PI3K C > T genotypes were associated with breast cancer predisposition in recessive model OR 6.96. Polymorphic variants of KLF-14 A > G, MDM4G > A, MiR-27aA >G, miR-196a-C > T were strongly associated with stage, tamoxifen treatment. Risk variants have been reported by whole exome sequencing in our BC patients. It was concluded that a strong association between the PI3K-AKT signaling pathway gene variants with the breast cancer susceptibility and progression. Similarly, KLF 14-AA, MDM4-GA, miR27a-GG and miR-196a-CT gene variants were associated with the higher risk probability of BC and were strongly correlated with staging of the BC patients. This study also reported Low, novel, and intermediate-genetic-risk variants of PI3K, AKT-1, MDM4G & KLF-14 by utilizing whole-exome sequencing. These variants should be further investigated in larger cohorts' studies.

Leflunomide an immunomodulator with antineoplastic and antiviral potentials but drug-induced liver injury: A comprehensive review.

Leflunomide (LF) represents the prototype member of dihydroorotate dehydrogenase (DHODH) enzyme inhibitors. DHODH is a mitochondrial inner membrane enzyme responsible for catalytic conversion of dihydroorotate into orotate, a rate-limiting step in the de novo synthesis of the pyrimidine nucleotides. LF produces cellular depletion of pyrimidine nucleotides required for cell growth and proliferation. Based on the affected cells the outcome can be attainable as immunosuppression, antiproliferative, and/or the recently gained attention of the antiviral potentials of LF and its new congeners. Also, protein tyrosine kinase inhibition is an additional mechanistic benefit of LF, which inhibits immunological events such as cellular expansion and immunoglobulin production with an enhanced release of immunosuppressant cytokines. LF is approved for the treatment of autoimmune arthritis of rheumatoid and psoriatic pathogenesis. Also, LF has been used off-label for the treatment of relapsing-remitting multiple sclerosis. However, LF antiviral activity is repurposed and under investigation with related compounds under a phase-I trial as a SARS CoV-2 antiviral in cases with COVID-19. Despite success in improving patients' mobility and reducing joint destruction, reported events of LF-induced liver injury necessitated regulatory precautions. LF should not be used in patients with hepatic impairment or in combination with drugs elaborating a burden on the liver without regular monitoring of liver enzymes and serum bilirubin as safety biomarkers. This study aims to review the pharmacological and safety profile of LF with a focus on the LF-induced hepatic injury from the perspective of pathophysiology and possible protective agents.

Metformin Protects From Rotenone-Induced Nigrostriatal Neuronal Death in Adult Mice by Activating AMPK-FOXO3 Signaling and Mitigation of Angiogenesis.

Parkinson's disease (PD) is a neurodegenerative disease that affects substantia nigra dopamine neurons. Many studies have documented the role of oxidative stress and angiogenesis in the pathogenesis of PD. Metformin (MTF) is an antidiabetic medication and AMP-activated protein kinase (AMPK) regulator that has shown antioxidant and antiangiogenic properties in many disorders. The aim of this study is to investigate the neuroprotective effect of MTF in a mouse model of rotenone-prompted PD with a highlight on its influence on the AMPK/forkhead box transcription factor O3 (FOXO3) pathway and striatal angiogenesis. In the running study, PD was induced in mice using repeated doses of rotenone and concomitantly treated with MTF 100 or 200 mg/kg/day for 18 days. Rotarod and pole tests were used to examine the animals' motor functionality. After that, animals were sacrificed, and brains were isolated and processed for immunohistochemical investigations or biochemical analyses. Oxidant stress and angiogenic markers were measured, including reduced glutathione, malondialdehyde, the nuclear factor erythroid 2-related factor 2 (Nrf2), hemoxygenase-1, thioredoxin, AMPK, FOXO3, and vascular endothelial growth factor (VEGF). Results indicated that MTF improved animals' motor function, improved striatal glutathione, Nrf2, hemoxygenase-1, and thioredoxin. Furthermore, MTF upregulated AMPK-FOXO3 proteins and reduced VEGF and cleaved caspase 3. MTF also increased the number of tyrosine hydroxylase (TH)-stained neurons in the substantia nigra neurons and in striatal neuronal terminals. This study is the first to highlight that the neuroprotective role of MTF is mediated through activation of AMPK-FOXO3 signaling and inhibition of the proangiogenic factor, VEGF. Further studies are warranted to confirm this mechanism in other models of PD and neurodegenerative diseases.

Opioid-induced hypogonadism: Pathophysiology, clinical and therapeutics review.

Opioids are pivotal therapeutics in the management of escalated chronic pain (moderate-severe). In the last two decades, the increased prescription rate and the prolonged usage of opioids shed light on opioid-induced endocrinopathy. Opioid-induced hypogonadism (OHG) results upon long-term opioid therapy. Clinically, patients with OHG are presented mainly by sexual dysfunction and infertility. Opioid clinical use in pain therapy is indispensable. However, the resultant sexual endocrinopathy cannot be overlooked and hence hormonal replacement therapy with regular monitoring of the patients represents a potential therapeutic strategy while avoiding opioids in patients with guaranteed long therapeutic exposure and switching to using low-dose naltrexone as alternative represents a possible prophylactic measure to ensure therapeutic compliance and secure a good life quality of patients.

The effect of low-dose naltrexone on solid Ehrlich carcinoma in mice: The role of OGFr, BCL2, and immune response.

AIMS: Cancer is a major worldwide health problem. Cancer cells express opioid growth factor (OGF) which controls their growth. Naltrexone in low dose (LDN) blocks opioid receptors intermittently and controls the replication of cancer cells. The aim of this study was to investigate the effect of LDN and its chemotherapeutic additive effect on the growth of solid Ehrlich carcinoma in mice with focus on the OGFr and immune responses. MAIN METHODS: Sixty female Swiss albino mice were assigned into 5 groups (n: 12 mice each): (i): normal control, (ii): Solid Ehrlich carcinoma (SEC), (iii): SEC treated with LDN, (iv): SEC treated with 5-fluorouracil (5-FU), (v): SEC treated with LDN + 5-FU. All drugs were started when the tumor became palpable on 9th day. At the end of the study animals were sacrificed, blood and tissue samples were collected. Tumor weight and volume were measured. Splenocytes and myeloid derived suppressor cells (MDSC) were counted. Tumor expression of opioid growth factor receptors (OGFr), serum level of IFN-γ, tumor histopathology (H&E) and immunohistochemistry staining of p21, p53, Bcl2 were assessed. KEY FINDINGS: All drug-treated groups showed reduction in tumor weight and volume, significant increase of splenocyte with tendency to reduce MDSC cell counts. LDN led to significant increase in OGFr both in solo and in combination with 5FU. Serum IFN-γ is significantly increased by LDN but decreased by 5-FU. Also, LDN and 5FU increased immunehistochemical staining of p21 while decreased immunostaining of Bcl2. In animals treated with a combination of LDN and 5FU a maximal downregulation of the antiapoptotic mediator BCL2 was observed. SIGNIFICANCE: The current study suggested that LDN may play a role in inhibiting cancer cell growth and highlights the possibility of promising combination with cancer chemotherapeutics, which guarantee further clinical studies for approval.

Carbamazepine Alleviates Retinal and Optic Nerve Neural Degeneration in Diabetic Mice via Nerve Growth Factor-Induced PI3K/Akt/mTOR Activation.

Aim: Diabetic retinopathy causes loss of vision in adults at working-age. Few therapeutic options are available for treatment of diabetic retinopathy. Carbamazepine (CARB), a widely used antiepileptic drug, was recently accounted for its neuroprotective effect. Nerve growth factor (NGF) activates various cascades among which, PI3K/Akt/mTOR pathway has a vital action in NGF-mediated neuronal differentiation and survival. This study evaluated the effect of CARB in the treatment of diabetic retina and unveiled some of the underlying molecular mechanisms. Main Methods: Alloxan diabetes model was induced in 36 albino well-acclimatized mice. After establishment of the diabetic model in 9 weeks, mice were assigned to treatment groups: (1) saline, (2) alloxan-diabetic, (3 and 4) alloxan+CARB (25 or 50 mg per kg p.o) for 4 weeks. After completion of the therapeutic period, mice were sacrificed and eyeballs were enucleated. Retinal levels of NGF and PI3K/Akt were assessed using real-time polymerase chain reaction. Further, total and phosphorylated TrKA, PI3K, Akt, mTOR as well as Caspase-3 were measured by Western blot analysis. Key Findings: Histopathological examination demonstrated that CARB attenuated vacuolization and restored normal thickness and organization of retinal cell layers. In addition, CARB increased pTrKA/TrKA ratio and ameliorated diabetes-induced reduction of NGF mRNA and immunostaining in retina. Additionally, it augmented the mRNA expression of PI3K and Akt, as well as the protein level of the phosphorylated PI3/Akt/mTOR. Significance: Results highlighted, for the first time, the neuronal protective effect for CARB in diabetic retina, which is mediated, at least in part, by activation of the NGF/PI3K/Akt/mTOR pathway.

Leflunomide-induced liver injury in mice: Involvement of TLR4 mediated activation of PI3K/mTOR/NFκB pathway.

AIMS: Leflunomide is a disease modifying anti-rheumatic drug (DMARD) beneficial in refractory cases of rheumatoid arthritis. Since leflunomide approval, hepatotoxicity and instructions of liver function monitoring have been recommended. The current work aimed to explore the possible role of inflammation in leflunomide-induced hepatotoxicity with a focus on the TLR4-mediated stimulation of PI3K/mTOR/NFκB pathway. MAIN METHODS: Forty-eight male albino mice were allocated into four different groups (n; 12 mice/group). Group (i): normal mice, Group (ii-iv) mice received escalating dosed/s of leflunomide (2.5, 5 or 10 mg/kg, p.o.) every 48 h for eight weeks. At the end of the study, mice were sacrificed, and blood samples were collected for determination of liver enzymes. Liver samples were collected; (1) formalin-fixed for histologic examination, (2) frozen for PI3K and mTOR genes PCR assays. KEY FINDINGS: Results indicated a significant elevation of liver enzymes in leflunomide-treated mice (10 mg/kg); AST and ALT activities were 218.17 ±â€¯6.83 U/L and 99.83 ±â€¯9.82 U/L versus 130.5 ±â€¯12.79 U/L and 44.72 ±â€¯3.58 U/L in the vehicle group. Additionally, histopathological examination revealed higher necro-inflammatory scores in leflunomide-treated mice. Immunohistochemistry indicated dose-dependent increased staining of TLR4 and caspase 3. Furthermore, leflunomide-treated mice (5 or 10 mg/kg) showed greater staining for NFκB compared to vehicle control. RT-PCR results revealed upregulations in genes expressing PI3K and mTOR by leflunomide. SIGNIFICANCE: The current study highlights the possible role of TLR4-PI3K/mTOR/NFκB in the pathogenesis of leflunomide-induced hepatic injury. A better understanding of mechanisms of leflunomide-induced hepatotoxicity may be of translational implication for the predictive, preventive and therapeutic purposes.

Neuroprotective effect of levetiracetam in mouse diabetic retinopathy: Effect on glucose transporter-1 and GAP43 expression.

AIMS: Retinopathy is a neurodegenerative complication associating diabetes mellitus. Diabetic retinopathy (DR) is the primary reason of visual loss during early adulthood. DR has a complicated multifactorial pathophysiology initiated by hyperglycaemia-induced ischaemic neurodegenerative retinal changes, followed by vision-threatening consequences. The main therapeutic modalities for DR involve invasive delivery of intravitreal antiangiogenic agents as well as surgical interventions. The current work aimed to explore the potential anti-inflammatory and retinal neuroprotective effects of levetiracetam. MAIN METHODS: This study was performed on alloxan-induced diabetes in mice (n: 21). After 10 weeks, a group of diabetic animals (n: 7) was treated with levetiracetam (25 mg/kg) for six weeks. Retinal tissues were dissected and paraffin-fixed for examination using (1) morphometric analysis with haematoxylin and eosin (HE), (2) immunohistochemistry (GLUT1, GFAP and GAP43), and (3) RT-PCR-detected expression of retinal inflammatory and apoptotic mediators (TNF-α, IL6, iNOS, NF-κB and Tp53). KEY FINDINGS: Diabetic mice developed disorganized and debilitated retinal layers with upregulation of the gliosis marker GFAP and downregulation of the neuronal plasticity marker GAP43. Additionally, diabetic retinae showed increased transcription of NF-κB, TNF-α, IL6, iNOS and Tp53. Levetiracetam-treated mice showed downregulation of retinal GLUT1 with relief and regression of retinal inflammation and improved retinal structural organization. SIGNIFICANCE: Levetiracetam may represent a potential neuroprotective agent in DR. The data presented herein supported an anti-inflammatory role of levetiracetam. However, further clinical studies may be warranted to confirm the effectiveness and safety of levetiracetam in DR patients.

Pharmacological Action of a Pregnane Glycoside, Russelioside B, in Dietary Obese Rats: Impact on Weight Gain and Energy Expenditure.

Background and purpose: Russelioside B (RB) is a pregnane glycoside obtained from Caralluma quadrangula; a herb with antidiabetic, anti-inflammatory, and antihyperlipidemic activities. The present experiment tested the possible role of RB in controlling weight gain in rats fed on high fat (HF) diet. Methods: RB was separated from the n-butanol fraction of the crude methanolic extract by chromatographic separation on a Si gel column according to the procedures described previously. The experiment of the biological assessment of RB used 32 male Wistar rats (4 groups, n = 8). Group 1 rats were fed with a palatable normal diet. Group 2, 3, and 4 were fed on HF diet for 16 weeks. Group 2 served as the HF diet control group while Group 3 and 4 received daily oral doses of RB (25 and 50 mg/kg) during the last four weeks. Animals' parameters like weight gain, fasting level of blood sugar, serum lipids, and serum liver enzyme activities were measured. Liver or adipose tissue weight was divided by the rat's body weight and multiplied by 100 to obtain the liver or adipose tissue index, respectively. Adipose tissues were processed for histopathological examination, measurement of mRNA expression of visfatin, leptin, adiponectin, uncoupling protein-1 (UCP-1), and carnitine palmitoyl transferase-1 (CPT-1). Furthermore, serum levels of insulin, interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), leptin, resistin, and adiponectin were assessed using ELISA kits. Results: Rats fed with the HF diet exhibited significant body weight gain, abnormal liver function, disturbed lipid profile, and greater serum level of pro-inflammatory cytokines in addition to greater insulin resistance, adipose tissue and liver indices. Further, rats fed with the HF diet displayed upregulations in the expression of visfatin and leptin with downregulations in the expression of adiponectin, UCP-1, and CPT-1 compared to normal rats. Interestingly, RB (25 or 50 mg/kg) favorably modulated the measured parameters. Conclusion: Data from this study documented the beneficial role of RB in diminishing weight gain, improving the inflammatory perturbations and energy expenditure in HF diet fed rats. Therefore, RB might be a promising candidate for obesity.

Mitigation of cadmium-induced lung injury by Nigella sativa oil.

Induction of oxidative stress and inflammation are considered the primary mechanism of cadmium (Cd) toxicity. Nigella sativa (NS) seeds and their oil (NSO) have been reported to possess antioxidant and anti-inflammatory potential. This study was conducted to assess the protective effect of NSO on Cd-induced lung damage in rat. Forty adult male Wistar rats were divided equally into 4 groups. Animals in groups I, II, and III received 1 ml of isotonic saline intraperitoneally (IP), 2 mg/kg of cadmium chloride (CdCl2) dissolved in isotonic saline IP, and 1 ml/kg of NSO by gastric gavage, respectively. Group IV rats received NSO an hour prior to CdCl2 administration via the same routes and doses as previously described. All animals were treated for 28 days. At the end of the study, animals were sacrificed; lungs were harvested for histopathological studies using light and electron microscopy. Saline-treated and NSO-treated rats showed normal lung parenchyma. However, CdCl2-treated rats showed massive degenerative changes in alveolar epithelial lining, disrupted interalveolar septa, and hemolytic debris in alveoli. Rats treated with both NSO and CdCl2 (group IV) showed amelioration of most Cd-induced lung damage with minimal histopathological changes in lung architecture. This study elucidates the protective effects of NSO on Cd-induced lung injury in rats and highlights the possibility of using NSO as a protective agent in individuals at high risk of Cd-induced lung toxicity.

ß-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis.

ß-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple ß-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple ß-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.

Characterization of the antigen specificity of T-cell clones from piperacillin-hypersensitive patients with cystic fibrosis.

ß-Lactam antibiotics provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but their use is limited by a high frequency of delayed-type allergic reactions. The objective of this study was to use cloned T-cells expressing a single T-cell receptor from five piperacillin-hypersensitive patients to characterize both the cellular pathophysiology of the reaction and antigen specificity to define the mechanism of activation of T-cells by piperacillin. More than 400 piperacillin-responsive CD4+, CD4+CD8+, or CD8+ T-cell clones were generated from lymphocyte transformation test and ELIspot-positive patients. The T-cell response (proliferation, T helper 2 cytokine secretion, and cytotoxicity) to piperacillin was concentration-dependent and highly specific. Enzyme-linked immunosorbent assay, gel electrophoresis, and mass spectrometry revealed that piperacillin bound exclusively to albumin in T-cell culture. Irreversible piperacillin binding at Lys 190, 195, 199, 432, and 541 on albumin and the stimulation of T-cells depended on incubation time. A synthetic piperacillin albumin conjugate stimulated T-cell receptors via a major histocompatibility complex- and processing-dependent pathway. Flucloxacillin competes for the same Lys residues on albumin as piperacillin, but the resulting conjugate does not stimulate T-cells, indicating that binding of the ß-lactam hapten in peptide conjugates confers structural specificity on the activation of the T-cell receptors expressed on drug-specific clones. Collectively, these data describe the cellular processes that underlie the structural specificity of piperacillin antigen binding in hypersensitive patients with cystic fibrosis.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis.

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

Trimethoprim stimulates T-cells through metabolism-dependent and -independent pathways.

Pathways of drug-specific T-cell stimulation have not been fully defined. The aim of this study was to use T-cell clones from a patient hypersensitive to the drug trimethoprim to characterize the involvement of drug metabolism and processing in antigen presentation and cross-reactivity patterns. The MHC-restricted CD4+ and CD8+ T-cell response was dependent on the presence of antigen-presenting cells, and both processing-dependent and -independent pathways of antigen presentation were detected. Stimulation of certain clones was blocked through inhibition of drug-metabolizing enzyme activity. Trimethoprim clones were additionally stimulated with diaveridine and pyrimethamine but not other closely related structures.

Enhanced antigenicity leads to altered immunogenicity in sulfamethoxazole-hypersensitive patients with cystic fibrosis.

BACKGROUND: Exposure of patients with cystic fibrosis to sulfonamides is associated with a high incidence of hypersensitivity reactions. OBJECTIVE: To compare mechanisms of antigen presentation and characterize the phenotype and function of T cells from sulfamethoxazole-hypersensitive patients with and without cystic fibrosis. METHODS: T cells were cloned from 6 patients and characterized in terms of phenotype and function. Antigen specificity and mechanisms of antigen presentation to specific clones were then explored. Antigen-presenting cell metabolism of sulfamethoxazole was quantified by ELISA. The involvement of metabolism in antigen presentation was evaluated by using enzyme inhibitors. RESULTS: Enzyme inhibitable sulfamethoxazole-derived protein adducts were detected in antigen-presenting cells from patients with and without cystic fibrosis. A significantly higher quantity of adducts were detected with cells from patients with cystic fibrosis. Over 500 CD4(+) or CD8(+) T-cell clones were generated and shown to proliferate and kill target cells. Three patterns of MHC-restricted reactivity (sulfamethoxazole-responsive, sulfamethoxazole metabolite-responsive, and cross-reactive) were observed with clones from patients without cystic fibrosis. From patients with cystic fibrosis, sulfamethoxazole metabolite-responsive and cross-reactive, but not sulfamethoxazole-responsive, clones were observed. The response of the cross-reactive clones to sulfamethoxazole was dependent on adduct formation and was blocked by glutathione and enzyme inhibitors. Antigen-stimulated clones from patients with cystic fibrosis secreted higher levels of IFN-γ, IL-6, and IL-10, but lower levels of IL-17. CONCLUSION: Sulfamethoxazole metabolism and protein adduct formation is critical for the stimulation of T cells from patients with cystic fibrosis. T cells from patients with cystic fibrosis secrete high levels of IFN-γ, IL-6, and IL-10.

Stimulation of human T cells with sulfonamides and sulfonamide metabolites.

BACKGROUND: Exposure to sulfonamides is associated with a high incidence of hypersensitivity reactions. Antigen-specific T cells are involved in the pathogenesis; however, the nature of the antigen interacting with specific T-cell receptors is not fully defined. OBJECTIVE: We sought to explore the frequency of sulfamethoxazole (SMX)- and SMX metabolite-specific T cells in hypersensitive patients, delineate the specificity of clones, define mechanisms of presentation, and explore additional reactivity with structurally related sulfonamide metabolites. METHODS: SMX- and SMX metabolite-specific T-cell clones were generated from 3 patients. Antigen specificity, mechanisms of antigen presentation, and cross-reactivity of specific clones were then explored. Low-lying energy conformations of drugs (metabolites) were modeled, and the energies available for protein binding was estimated. RESULTS: Lymphocytes proliferated with parent drugs (SMX, sulfadiazine, and sulfapyridine) and both hydroxylamine and nitroso metabolites. Three patterns of drug (metabolite) stimulation were seen: 44% were SMX metabolite specific, 43% were stimulated with SMX metabolites and SMX, and 14% were stimulated with SMX alone. Most metabolite-responsive T cells were stimulated with nitroso SMX-modified protein through a hapten mechanism involving processing. In contrast to SMX-responsive clones, which were highly specific, greater than 50% of nitroso SMX-specific clones were stimulated with nitroso metabolites of sulfapyridine and sulfadiazine but not nitrosobenzene. Pharmacophore modeling showed that the summation of available binding energies for protein interactions and the preferred spatial arrangement of atoms in each molecule determine a drug's potential to stimulate specific T cells. CONCLUSIONS: Nitroso sulfonamide metabolites form potent antigenic determinants for T cells from hypersensitive patients. T-cell responses against drugs (metabolites) bound directly to MHC or MHC/peptide complexes can occur through cross-reactivity with the haptenic immunogen.