Management of Lobular Neoplasia Diagnosed by Core Biopsy.

Lobular neoplasia (LN) involves proliferative changes within the breast lobules. LN is divided into lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH). LCIS can be further subdivided into three subtypes: classic LCIS, pleomorphic LCIS, and LCIS with necrosis (florid type). Because classic LCIS is now considered as a benign etiology, current guidelines recommend close follow-up with imaging versus surgical excision. The goal of our study was to determine if the diagnosis of classic LN on core needle biopsy (CNB) merits surgical excision. This is a retrospective, observational study conducted at Mount Auburn Hospital, Cambridge, MA, from May 17, 2017, through June 30, 2020. We reviewed the data of breast biopsies conducted at our hospital over this period and included patients who were diagnosed with classic LN (LCIS and/or ALH) and excluded patients having any other atypical lesions on CNB. All known cancer patients were excluded. Of the 2707 CNBs performed during the study period, we identified 68 women who were diagnosed with ALH or LCIS on CNB. CNB was performed for an abnormal mammogram in the majority of patients (60; 88%) while 7(10.3%) had an abnormal breast magnetic resonance imaging study (MRI), and 1 had an abnormal ultrasound (US). A total of 58 patients (85%) underwent excisional biopsy, of which 3 (5.2%) showed malignancy, including 2 cases of DCIS and 1 invasive carcinoma. In addition, there was 1 case (1.7%) with pleomorphic LCIS and 11 cases with ADH (15.5%). The management of LN found on core biopsy is evolving, with some advocating surgical excision and others recommending observation. Our data show a change in diagnosis with excisional biopsy in 13 (22.4%) of patients with 2 cases of DCIS, 1 invasive carcinoma, 1 pleomorphic LCIS, and 9 cases of ADH, diagnosed on excisional biopsy. While ALH and classic LCIS are considered benign, the choice of ongoing surveillance versus excisional biopsy should be made with shared decision making with the patient, with consideration of personal and family history, as well as patient preferences.

Sleepless and Spent in Survivorship: Fatigue and Insomnia in Breast Cancer Survivors.

BACKGROUND: It is estimated that there are 3.8 million breast cancer survivors in the United States. Addressing survivors' post-treatment needs is critical to providing quality healthcare. METHODS: A standardized questionnaire for breast cancer survivors was employed to assess the health status, challenges, and concerns of our breast cancer patients at their survivorship visits, which were conducted 4 months after surgery. All patients were seen in the breast center at one community hospital over a 6-year period. RESULTS: Responses to a standardized questionnaire that was administered to 505 consecutive breast cancer patients at their survivorship visits 4 months after surgery were evaluated. The most striking finding was that 35% reported symptoms of insomnia, 26% had persistent fatigue, and 19% experienced fatigue that interfered with their usual activities. There was a significant association between symptoms of insomnia and radiation treatment (P = .004), pain (P < .001), hormone therapy (P < .01), and side effects of hormone therapy (P < .0001). There was also a significant association between fatigue and pain (P < .001) as well as side effects from hormone treatment (P = .0036). CONCLUSIONS: Over a third (35%) of breast cancer patients suffer from insomnia, while over a quarter (26%) complain of fatigue at their survivorship assessments. Contributing factors include radiation treatment, pain, and hormonal therapy. Careful assessment and treatment of fatigue and symptoms of insomnia in breast cancer patients is needed to improve quality of life for survivors.

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.

Follistatin-288-Fc Fusion Protein Promotes Localized Growth of Skeletal Muscle.

Follistatin is an endogenous glycoprotein that promotes growth and repair of skeletal muscle by sequestering inhibitory ligands of the transforming growth factor-ß superfamily and may therefore have therapeutic potential for neuromuscular diseases. Here, we sought to determine the suitability of a newly engineered follistatin fusion protein (FST288-Fc) to promote localized, rather than systemic, growth of skeletal muscle by capitalizing on the intrinsic heparin-binding ability of the follistatin-288 isoform. As determined by surface plasmon resonance and cell-based assays, FST288-Fc binds to activin A, activin B, myostatin (growth differentiation factor GDF8), and GDF11 with high affinity and neutralizes their activity in vitro. Intramuscular administration of FST288-Fc in mice induced robust, dose-dependent growth of the targeted muscle but not of surrounding or contralateral muscles, in contrast to the systemic effects of a locally administered fusion protein incorporating activin receptor type IIB (ActRIIB-Fc). Furthermore, systemic administration of FST288-Fc in mice did not alter muscle mass or body composition as determined by NMR, which again contrasts with the pronounced systemic activity of ActRIIB-Fc when administered by the same route. Subsequent analysis revealed that FST288-Fc in the circulation undergoes rapid proteolysis, thereby restricting its activity to individual muscles targeted by intramuscular administration. These results indicate that FST288-Fc can produce localized growth of skeletal muscle in a targeted manner with reduced potential for undesirable systemic effects. Thus, FST288-Fc and similar agents may be beneficial in the treatment of disorders with muscle atrophy that is focal, asymmetric, or otherwise heterogeneous.

Suppression of cancer relapse and metastasis by inhibiting cancer stemness.

Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity.

The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.

Helicobacter pylori induces up-regulation of the epidermal growth factor receptor in AGS gastric epithelial cells.

BACKGROUND: Helicobacter pylori infection increases the risk of gastric carcinogenesis. The aim of the present study was to determine whether H. pylori could up-regulate the expression of the epidermal growth factor receptor (EGFR), a critical gene in the carcinogenic process. METHODS: AGS gastric epithelial cells were infected with cag(+) toxigenic or cag(-) nontoxigenic strains of H. pylori or isogenic mutants. EGFR protein expression was determined by Western blotting and immunofluorescence. EGFR mRNA levels were evaluated using real-time polymerase chain reaction. The signaling pathways leading to EGFR up-regulation were examined using the ERK1/2 inhibitor PD98059, the Src inhibitor pp2, the nuclear factor- kappa B inhibitor caffeic acid phenethyl ester, EGFR neutralizing antibodies, and the EGFR kinase inhibitor AG1478. RESULTS: Infection of AGS cells by H. pylori significantly increased EGFR mRNA and protein levels. We found that this effect was limited to cag(+) H. pylori strains and that mutants with a defective type IV secretion system were unable to cause EGFR up-regulation. Increased EGFR expression was found to be dependent on EGFR receptor transactivation, ERK1/2 phosphorylation, and Src activation. CONCLUSION: Infection of gastric epithelial cells by H. pylori triggers an autocrine loop whereby EGFR transactivation leads to the up-regulation of EGFR expression. This, in turn, may contribute to unrestrained epithelial cell proliferation and carcinogenesis.

Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin.

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.

Confocal light absorption and scattering spectroscopic microscopy.

We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.

MIP-3alpha neutralizing monoclonal antibody protects against TNBS-induced colonic injury and inflammation in mice.

A characteristic feature of human inflammatory bowel disease, particularly Crohn's disease, is the presence of activated CD4(+) T cells. Recently, we have shown that colonic epithelial cell production of macrophage inflammatory protein (MIP)-3alpha, a CD4 T cell-directed chemokine, is elevated in inflammatory bowel disease. However, the functional relevance of MIP-3alpha production during intestinal inflammation is poorly understood. The aim of this study was to determine whether MIP-3alpha production is increased during murine 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and to examine the effect of anti-MIP-3alpha neutralizing monoclonal antibody administration in this model. We found that the administration of TNBS significantly increased colonic MIP-3alpha protein levels in Balb/c mice. Consistent with this, a marked increase in the number of CCR6-bearing lamina propria CD4(+) and CD8(+) T cells was also observed in TNBS-treated animals. Treatment of mice with an anti-MIP-3alpha neutralizing monoclonal antibody significantly reduced TNBS-mediated increases in colonic weight-to-length ratio, mucosal ulceration, histological damage, and myeloperoxidase activity. TNBS-mediated increases in the number of CCR6-bearing lamina propria T cells were also substantially reduced by anti-MIP-3alpha neutralizing monoclonal antibody treatment. Taken together, our findings indicate that blockade of MIP-3alpha bioactivity can significantly reduce TNBS-mediated colonic injury and T cell recruitment, suggesting a role for this chemokine in the pathophysiology of intestinal inflammation.

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression.

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.

Clostridium difficile toxoid vaccine in recurrent C. difficile-associated diarrhea.

BACKGROUND & AIMS: Recurrent C difficile -associated diarrhea (CDAD) is associated with a lack of protective immunity to C difficile toxins. A parenteral C difficile vaccine containing toxoid A and toxoid B was reported previously to be safe and immunogenic in healthy volunteers. Our aim was to examine whether the vaccine is also well tolerated and immunogenic in patients with recurrent CDAD. METHODS: Subjects received 4, 50-microg intramuscular inoculations of the C difficile vaccine over an 8-week period. Serum antitoxin antibodies were measured by ELISA, and toxin neutralizing activity was evaluated using the tissue culture cytotoxin assay. RESULTS: Three patients with multiple episodes of recurrent CDAD were vaccinated. Two of the 3 showed an increase in serum IgG antitoxin A antibodies (3-fold and 4-fold increases, respectively) and in serum IgG antitoxin B antibodies (52-fold and 20-fold, respectively). Both also developed cytotoxin neutralizing activity against toxin A and toxin B. Prior to vaccination, the subjects had required nearly continuous treatment with oral vancomycin for 7, 9, and 22 months, respectively, to treat recurrent episodes of CDAD. After vaccination, all 3 subjects discontinued treatment with oral vancomycin without any further recurrence. CONCLUSIONS: A C difficile toxoid vaccine induced immune responses to toxins A and B in patients with CDAD and was associated with resolution of recurrent diarrhea. The results of this study support the feasibility of active vaccination against C difficile and its toxins in high-risk individuals but must be validated in larger, randomized, controlled trials.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Update on the immunologic basis of Helicobacter pylori gastritis.

PURPOSE OF REVIEW: Helicobacter pylori is a ubiquitous bacterial pathogen that has evolved to chronically infect the gastric mucosal surface, evade host immune clearance, and cause peptic ulcer disease or gastric neoplasia in a significant minority of infected individuals. Understanding the colonization, persistence, and virulence determinants of the bacterium as well as the innate and adaptive immune responses of the host are critically important if we are to develop novel treatment strategies for eradication of infection and prevention of H. pylori-induced gastroduodenal disease. RECENT FINDINGS: Substantial progress has been made in understanding the role of CagA in altering gastric epithelial cell signaling pathways. Intracellular CagA has been shown to activate the Ras/MEK/ERK mitogen activated protein kinase cascade and to associate with epithelial tight-junction scaffolding protein ZO-1. CagA was shown to regulate cellular responses and possibly contribute to gastritis by phosphorylation-dependent pathways. A phosphorylation-independent mechanism for CagA intracellular effects has also been proposed. The potential for CagA to disrupt the apical junctional barrier and for the outer membrane protein oipA to promote IL-8 secretion and gastric inflammation have also been explored. A number of different mechanisms by which H. pylori escapes and evades host immune attack to cause chronic indolent inflammation have been uncovered. Meanwhile, the examination and development of new adjuvants and vaccine delivery mechanisms to induce mucosal immune responses against key bacterial antigens has been a continuing focus of investigation in both animal and human studies. SUMMARY: H. pylori induces gastritis through production of a variety of antigens, virulence factors, and soluble mediators. The bacterium also dysregulates, disarms, and evades host immune responses to maintain chronic colonization of the gastric mucosa. Understanding the mechanisms of its growth and survival in the human stomach are essential for the development of an effective vaccine and other novel eradication strategies.

Altered gene expression in three plant species in response to treatment with Nep1, a fungal protein that causes necrosis.

Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasive weed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 micro g mL(-)(1), 0.1% [v/v] Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Taraxacum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment. Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolate cDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential gene expression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knapweed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. In addition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12) of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein (AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated (5 micro g mL(-)(1), 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spotted knapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levels of mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-L77-treated plants between 15 min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 micro g mL(-)(1), 0.1% [v/v] Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1 in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved or are related to gene families that are involved in plant stress responses, including wounding, drought, senescence, and disease resistance.

ESE-1, an enterocyte-specific Ets transcription factor, regulates MIP-3alpha gene expression in Caco-2 human colonic epithelial cells.

We have previously shown that colonic epithelial cells are a major site of MIP-3alpha production in human colon and that enterocyte MIP-3alpha protein levels are elevated in inflammatory bowel disease. The aim of this study was to determine the molecular mechanisms regulating MIP-3alpha gene transcription in Caco-2 intestinal epithelial cells. We show that a kappaB element at nucleotides -82 to -93 of the MIP-3alpha promoter binds p50/p65 NF-kappaB heterodimers and is a major regulator of basal and interleukin-1beta (IL-1beta)-mediated gene activation. Scanning mutagenesis of the MIP-3alpha 5'-flanking region also identified two additional binding elements: Site X (nucleotides -63 to -69) and Site Y (nucleotides -143 to -154). Site X (CGCCTTC) bound Sp1 and regulated basal MIP-3alpha gene transcription. Overexpression of Sp1 increased basal luciferase activity, whereas, substitutions in the Sp1 element significantly reduced reporter activity. In contrast, Site Y (AAGCAGGAAGTT) regulated both basal and cytokine-induced gene activation and bound the Ets nuclear factor ESE-1. Substitutions in the Site Y element markedly reduced inducible MIP-3alpha reporter activity. Conversely, overexpression of ESE-1 significantly up-regulated MIP-3alpha luciferase levels. Taken together, our findings demonstrate that co-ordinate activation and binding of ESE-1, Sp1, and NF-kappaB to the MIP-3alpha promoter is required for maximal gene expression by cytokine-stimulated Caco-2 human intestinal epithelial cells.

Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation.

BACKGROUND & AIMS: Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor kappaB (NF-kappaB) activation and interleukin (IL)-8 release from toxin A-exposed enterocytes. METHODS: NF-kappaB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function. RESULTS: HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P < 0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IkappaB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-kappaB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1beta was not affected. CONCLUSIONS: Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-kappaB before release of IL-8.