UBXN2A suppresses the Rictor-mTORC2 signaling pathway, an established tumorigenic pathway in human colorectal cancer.

The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.

Pre-clinical safety and therapeutic efficacy of a plant-based alkaloid in a human colon cancer xenograft model.

A high-throughput drug screen revealed that veratridine (VTD), a natural plant alkaloid, induces expression of the anti-cancer protein UBXN2A in colon cancer cells. UBXN2A suppresses mortalin, a heat shock protein, with dominant roles in cancer development including epithelial-mesenchymal transition (EMT), cancer cell stemness, drug resistance, and apoptosis. VTD-dependent expression of UBXN2A leads to the deactivation of mortalin in colon cancer cells, making VTD a potential targeted therapy in malignant tumors with high levels of mortalin. VTD was used clinically for the treatment of hypertension in decades past. However, the discovery of newer antihypertensive drugs and concerns over potential neuro- and cardiotoxicity ended the use of VTD for this purpose. The current study aims to determine the safety and efficacy of VTD at doses sufficient to induce UBXN2A expression in a mouse model. A set of flow-cytometry experiments confirmed that VTD induces both early and late apoptosis in a dose-dependent manner. In vivo intraperitoneal (IP) administration of VTD at 0.1 mg/kg every other day (QOD) for 4 weeks effectively induced expression of UBXN2A in the small and large intestines of mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays on tissues collected from VTD-treated animals demonstrated VTD concentrations in the low pg/mg range. To address concerns regarding neuro- and cardiotoxicity, a comprehensive set of behavioral and cardiovascular assessments performed on C57BL/6NHsd mice revealed that VTD generates no detectable neurotoxicity or cardiotoxicity in animals receiving 0.1 mg/kg VTD QOD for 30 days. Finally, mouse xenograft experiments in athymic nude mice showed that VTD can suppress tumor growth. The main causes for the failure of experimental oncologic drug candidates are lack of sufficient safety and efficacy. The results achieved in this study support the potential utility of VTD as a safe and efficacious anti-cancer molecule.

Hemodynamic patterns associated with activation of bradykinin-sensitive pericardial afferents.

The heart is endowed with reflexogenic areas capable of powerful blood pressure responses. Relatively little work has studied the hemodynamic mechanisms underlying these responses and whether these are sexually dimorphic. We hypothesized that activation of bradykinin-sensitive pericardial afferents would produce a sexually dimorphic cardiac output response. Male and female Sprague Dawley rats were anesthetized and instrumented with catheters for recording arterial pressure, with an aortic arch flow probe to record cardiac output and with a catheter in the pericardial sac. Mean arterial pressure (MAP), cardiac index (CI) and total peripheral resistance index (TPRI) responses to pericardial bradykinin injection (0.1, 1 µg/kg) were recorded. Pericardial bradykinin injection caused similar increases in MAP in male and female rats. However, the underlying hemodynamic patterns varied considerably. We identified a cluster of CI responders and TPRI responders in both male and female rats. Within CI responders, females exhibited greater CI increases than males. Conversely, in TPRI responders, males exhibited a greater TPRI increase than females. We conclude that aggregate activation of bradykinin-sensitive pericardial afferents is associated with a relatively uniform pressor response but different hemodynamic patterns with males exhibiting a more robust vascular response and females a more robust cardiac output response.

Myocardial contrast echocardiography assessment of mouse myocardial infarction: comparison of kinetic parameters with conventional methods.

This study explores the use of a minimally invasive assessment of myocardial infarction (MI) in mice using myocardial contrast echocardiography (MCE). The technique uses existing equipment and software readily available to the average researcher. C57/BL6 mice were randomized to either MI or sham surgery and evaluated using MCE at 1- or 2-weeks post-surgery. Size-isolated microbubbles were injected via retro-orbital catheter where their non-linear characteristics were utilized to produce the two-dimensional parameters of Wash-in-Rate and the Peak Enhancement, indicative of relative myocardial perfusion and blood volume, respectively. Three-dimensional cardiac reconstructions allowed the calculation of the Percent Agent, interpreted as the vascularity of the entire myocardium. These MCE parameters were compared to conventional assessments including M-Mode, strain analysis, and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Except for the Wash-in-Rate 2-week cohort, all MCE parameters were able to differentiate sham-operated versus MI animals and correlated with TTC staining (P < 0.05). MCE parameters were also able to identify MI group animals which failed to develop infarctions as determined by TTC staining. This study provides basic validation of these MCE parameters to detect MI in mice complementary to conventional methods while providing additional hemodynamic information in vivo.

The emergence of cardiac changes following the self-administration of methamphetamine.

BACKGROUND: Clinical observations suggest an association between methamphetamine (METH) use and cardiovascular disease, but preclinical studies are lacking. The purpose of the current study was to explore changes in left ventricular function as a potential precursor to cardiovascular disease in a rodent model of METH use. METHODS: Male rats were allowed to self-administer either METH or saline for 9 d. On the day following the 4th and 9th self-administration sessions, an echocardiogram was performed to assess left-ventricular parameters under basal conditions and following a low-dose of METH (1 mg/kg). RESULTS: A low challenge dose of METH resulted in subtle but statistically significant changes in cardiac function during the echocardiogram in both the METH and saline self-administering groups. Further, differences in left-ventricular parameters such as stroke volume and heart rate were observed between METH and saline groups following the 9th self-administration session. Finally, supervised machine learning correctly predicted the self-administration group assignment (saline or METH) using cardiac parameters following the 9th self-administration session. CONCLUSIONS: The findings of the current study suggest the heart, specifically the left ventricle, is sensitive to METH. Overall, these findings and emerging clinical observations highlight the need for research to investigate the effects of METH use on the heart.

Activation of bradykinin-sensitive pericardial afferents increases systemic venous tone in conscious rats.

Our understanding of reflex regulation of veins lags behind that of the arterial system. While the cardiac sympathetic afferent reflex (CSAR) exerts control over sympathetic outflow, its effect on venous tone is not known. We tested the hypothesis that activation of pericardial bradykinin sensitive afferents elicits systemic venoconstriction. Male and female Sprague Dawley rats were chronically instrumented for measurement of arterial pressure and mean circulatory filling pressure, an index of venous tone, and with an indwelling pericardial catheter. Mean arterial pressure, heart rate and mean circulatory filling pressure responses were assessed in conscious rats in response to graded pericardial injections of bradykinin (1.5-20 µg/kg) before and after ganglionic blockade, and to intravenous norepinephrine (0.05-0.8 µg/kg). Bradykinin B2 receptor was assessed by Western blot. Pericardial bradykinin injections caused graded increases in mean arterial pressure, heart rate and mean circulatory filling pressure. These responses were markedly attenuated after autonomic blockade. The increments in mean circulatory filling pressure were attenuated in female rats. There were no differences in the venoconstrictor responses to norepinephrine or ventricular bradykinin receptor expression between male and females. We interpret these findings to indicate that activation of bradykinin sensitive pericardial afferents elicits a sexually dimorphic, autonomically mediated systemic venoconstrictor response. Differences in venous smooth muscle responses to norepinephrine or ventricular bradykinin receptor expression do not account for the sexual dimorphism. We conclude that systemic venoconstriction contributes to the overall hemodynamic response to activation of the cardiac sympathetic afferent reflex and that this effect is sexually dimorphic.

UCHL1 regulates muscle fibers and mTORC1 activity in skeletal muscle.

AIMS: Skeletal muscle wasting is associated with many chronic diseases. Effective prevention and treatment of muscle wasting remain as a challenging task due to incomplete understanding of mechanisms by which muscle mass is maintained and regulated. This study investigated the functional role of Ubiquitin C-terminal hydrolase L1 (UCHL1) in skeletal muscle. MAIN METHODS: Mice with skeletal muscle specific gene knockout of UCHL1 and C2C12 myoblast cells with UCHL1 knockdown were used. Muscle fiber types and size were measured using tissue or cell staining. The mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 activities were assessed with the phosphorylation of their downstream targets. KEY FINDINGS: In mouse skeletal muscle, UCHL1 was primarily expressed in slow twitch muscle fibers. Mice with skeletal muscle specific knockout (skmKO) of UCHL1 exhibited enlarged muscle fiber sizes in slow twitch soleus but not fast twitch extensor digitorum longus (EDL) muscle. Meanwhile, UCHL1 skmKO enhanced mTORC1 activity and reduced mTORC2 activity in soleus but not in EDL. Consistently, in C2C12 cells, UCHL1 knockdown increased the myotube size, enhanced mTORC1 activity, and reduced mTORC2 activities as compared with control cells. UCHL1 knockdown did not change the major proteins of mTOR complex but decreased the protein turnover of PRAS40, an inhibitory factor of mTORC1. SIGNIFICANCE: These data revealed a novel function of UCHL1 in regulation of mTORC1 activity and skeletal muscle growth in slow twitch skeletal muscle. Given the upregulation of UCHL1 in denervation and spinal muscle atrophy, our finding advances understanding of regulators that are involved in muscle wasting.

USP14 inhibitor attenuates cerebral ischemia/reperfusion-induced neuronal injury in mice.

Stroke is associated with over-production of misfolded and aggregating proteins. However, it remains largely unclear whether enhanced removal of protein aggregates following ischemic stroke is neuroprotective. Deubiquitinating enzymes (DUBs) are a large group of proteases that regulate protein degradation. The ubiquitin-specific protease 14 (USP14) is a DUB that is associated with the proteasome and negatively regulates proteasome activity. In this study, we examined the effect of 1-[1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl]-2-pyrrolidin-1-ylethanone (IU1), a specific small molecule inhibitor of USP14, on mouse focal cerebral ischemic stroke-induced neuronal injury in mice. We found that IU1 treatment attenuated ischemic stroke-caused neuronal injury, which was reflected by increased survival rate, reduced infarct volume, as well as decreased neuronal loss in the IU1-treated mice compared to the control-treated mice. Additionally, IU1 treatment is associated with reduced protein aggregates and enhanced proteasome functionality. These data not only highlight the significance of protein homeostasis in cerebral ischemia/reperfusion-induced neuronal injury but also extend the therapeutic role of DUB inhibitors.

Assessment of murine colorectal cancer by micro-ultrasound using three dimensional reconstruction and non-linear contrast imaging.

The relatively low success rates of current colorectal cancer (CRC) therapies have led investigators to search for more specific treatments. Vertebrate models of colorectal cancer are essential tools for the verification of new therapeutic avenues such as gene therapy. The evaluation of colorectal cancer in mouse models has been limited due to the lack of an accurate quantitative and longitudinal noninvasive method. This work introduces a method of three-dimensional micro-ultrasound reconstruction and microbubble administration for the comprehensive and longitudinal evaluation of CRC progression. This approach enabled quantification of both tumor volume and relative vascularity using a well-established inducible murine model of colon carcinogenesis. This inducible model recapitulated the adenocarcinoma sequence that occurs in human CRC allowing systematic in situ evaluation of the ultrasound technique. The administration of intravenous microbubbles facilitated enhancement of colon vascular contrast and quantification of relative vascularity of the mid and distal colon of the mouse in three dimensions. In addition, two-dimensional imaging in the sagittal orientation of the colon using Non-Linear Contrast Mode enabled calculation of relative blood volume and perfusion as the microbubbles entered the colon microvasculature. Quantitative results provided by the outlined protocol represent a noninvasive tool that can more accurately define CRC development and progression. This ultrasound technique will allow the practical and economical longitudinal study of murine CRC in both basic and preclinical studies.

Nucleocytoplasmic Translocation of UBXN2A Is Required for Apoptosis during DNA Damage Stresses in Colon Cancer Cells.

The subcellular localization, expression level, and activity of anti-cancer proteins alter in response to intrinsic and extrinsic cellular stresses to reverse tumor progression. The purpose of this study is to determine whether UBXN2A, an activator of the p53 tumor suppressor protein, has different subcellular compartmentalization in response to the stress of DNA damage. We measured trafficking of the UBXN2A protein in response to two different DNA damage stresses, UVB irradiation and the genotoxic agent Etoposide, in colon cancer cell lines. Using a cytosol-nuclear fractionation technique followed by western blot and immunofluorescence staining, we monitored and quantitated UBXN2A and p53 proteins as well as p53's downstream apoptotic pathway. We showed that the anti-cancer protein UBXN2A acts in the early phase of cell response to two different DNA damage stresses, being induced to translocate into the cytoplasm in a dose- and time-dependent manner. UVB-induced cytoplasmic UBXN2A binds to mortalin-2 (mot-2), a known oncoprotein in colon tumors. UVB-dependent upregulation of UBXN2A in the cytoplasm decreases p53 binding to mot-2 and activates apoptotic events in colon cancer cells. In contrast, the shRNA-mediated depletion of UBXN2A leads to significant reduction in apoptosis in colon cancer cells exposed to UVB and Etoposide. Leptomycin B (LMB), which was able to block UBXN2A nuclear export following Etoposide treatment, sustained p53-mot-2 interaction and had partially antagonistic effects with Etoposide on cell apoptosis. The present study shows that nucleocytoplasmic translocation of UBXN2A in response to stresses is necessary for its anti-cancer function in the cytoplasm. In addition, LMB-dependent suppression of UBXN2A's translocation to the cytoplasm upon stress allows the presence of an active mot-2 oncoprotein in the cytoplasm, resulting in p53 sequestration as well as activation of other mot-2-dependent growth promoting pathways.

A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2.

Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

Age-related attenuation of parasympathetic control of the heart in mice.

The autonomic nervous system maintains homeostasis through the balance of the sympathetic nervous system (SNS) and parasympathetic nervous system (PSNS). Especially evident in the heart, maintenance of this balance is important for the control of heart rate, conduction, and contractility. It is known that aging, similar to various cardiovascular diseases, results in an increase in SNS activity and a decrease in PSNS activity, which may contribute to age-related cardiac dysfunction and remodeling. Intracardiac ganglia relay and integrate the PSNS signals to the heart. Therefore, this study investigated whether altered function of intracardiac ganglia is involved in age-related parasympathetic dysfunction and the potential role of the major cholinergic components of intracardiac ganglionic transmission in the process. This study utilized two age groups of mice, the younger mice at 1-2.5 months of age, and the older mice at 11-12 months of age. The results show that the older mice exhibit diminishment of both baroreflex sensitivity and response to rostral-severed vagal stimulation but preserved response to administration of muscarinic acetylcholine receptor agonist, bethanechol. Analysis of whole atrial lysate revealed significant diminishments in choline acetyltransferase (ChAT) and the upper band of vesicular acetylcholine transporter (VAchT). In contrast, the upper band of the high affinity choline transporter (CHT) was significantly upregulated in the older group. Further analysis showed that the soluble but not insoluble fraction of CHT protein is significantly increased in the older group. This implicates a potential reduction of acetylcholine synthesis and/or release and an improper compensatory change of CHT may be responsible for the PSNS dysfunction exhibited in this model.

Cytisine induces autonomic cardiovascular responses via activations of different nicotinic receptors.

Nicotinic cholinergic receptors mediate autonomic transmission at ganglia. However, whether different subtypes of nicotinic cholinergic receptors expressed in autonomic ganglia elicit distinct roles in mediating sympathetic and parasympathetic regulations remain to be defined. In this study, we observed that different subtypes of nicotinic receptors were responsible for the sympathetic and parasympathetic cardiovascular responses. In urethane anesthetized mice, intravenous injection with cytisine, a non-selective nicotinic agonist, induced a brief but pronounced decrease in heart rate, followed by increases in heart rate and arterial blood pressure. The bradycardic response was blocked by atropine, and the pressor response was blocked by prazosin, confirming that these responses were parasympathetic and sympathetic activities, respectively. Hexamethonium, a ganglionic blocker, blocked both sympathetic and parasympathetic responses. Pretreatment with methyllycaconitine citrate, a selective alpha7 nicotinic receptor antagonist, significantly attenuated cytisine-induced sympathetic response with little effect on the parasympathetic response. In contrast, pretreatment with dihydro-beta-erythroidine hydrobromide, a selective alpha4beta2 nicotinic receptor antagonist, blocked cytisine-induced parasympathetic response but not the sympathetic response. Pretreatment with dihydro-beta-erythroidine hydrobromide also blocked baroreflex associated parasympathetic bradycardic response. Moreover, treatment with nicotine induced a bradycardic response without a significant pressor response, which was also attenuated by dihydro-beta-erythroidine hydrobromide. Collectively, these data suggest that different nicotinic receptors play distinct roles in sympathetic and parasympathetic ganglia. Specifically, activations of alpha7 and alpha4beta2 nicotinic receptors are involved in cytisine-induced cardiovascular sympathetic and parasympathetic responses, respectively.

Specific subtypes of nicotinic cholinergic receptors involved in sympathetic and parasympathetic cardiovascular responses.

Various subtypes of nicotinic cholinergic receptors are expressed in autonomic ganglia. The distinct functional roles of these receptors in autonomic ganglionic transmission to different target organs remain to be elucidated. In this study, we tested the sympathetic and parasympathetic cardiovascular responses to nicotinic agonist and antagonists in urethane-anesthetized mice. Intravenous injection with a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, induced a brief but pronounced decrease in heart rate, followed by significant increases in heart rate and arterial blood pressure. The bradycardic response was blocked by atropine whereas the pressor response was blocked by prazosine, confirming those responses were parasympathetic and sympathetic activities, respectively. The sympathetic response was blocked by methyllycaconitine citrate, a selective alpha 7 nicotinic cholinergic receptor (nAchR) antagonist. The parasympathetic response was blocked by a selective alpha 4 beta 2 nAchR antagonist, dihydro-beta-erythroidine hydrobromide. Moreover, injection with a selective alpha 4 beta 2 nAchR agonist, RJR2403 oxalate, induced a pronounced parasympathetic response with a smaller sympathetic response. Collectively, these data show that activations of alpha 4 beta 2 nAchRs elicits a parasympathetic cardiovascular response and activation of alpha 7 nAchRs elicits a sympathetic cardiovascular response. These data suggest that specific subtypes of nicotinic receptors at the level of the ganglia may play distinct roles in mediating sympathetic or parasympathetic activation.

Neostigmine and pilocarpine attenuated tumour necrosis factor alpha expression and cardiac hypertrophy in the heart with pressure overload.

The inflammatory cytokine tumour necrosis factor alpha (TNF alpha) is known to be a major factor contributing to cardiac remodelling and dysfunction. Parasympathetic nervous system cholinergic function can inhibit TNF alpha expression during systemic infection. In the present study, we tested the effects of a cholinesterase inhibitor, neostigmine, and a muscarinic cholinergic agonist, pilocarpine, on cardiac hypertrophy and TNF alpha levels during pressure overload. Rats with transverse aortic constriction exhibited elevated TNF alpha protein levels in the heart, increased heart weight to body weight ratios (an index of cardiac hypertrophy) and decreased left ventricular diastolic function. Two weeks of infusion with neostigmine (6 microg kg(-1) day(-1)) or pilocarpine (0.3 mg kg(-1) day(-1)) significantly reduced cardiac hypertrophy, reduced TNF alpha levels and elevated interleukin-10 levels in heart tissues, and improved ventricular function in rats with transverse aortic constriction. Neither of these treatments significantly changed ventricular pressure load. Furthermore, in primary cultured neonatal cardiac cells, treatment with pilocarpine attenuated adrenergic agonist phenylephrine-induced increased TNF alpha expression and [3H]leucine (a marker of protein synthesis) incorporation in the cells. Collectively, both cholinergic agents decreased TNF alpha levels and attenuated cardiac hypertrophy. Since both agents potentially enhanced cholinergic function, the anti-inflammatory action may be involved in the cardioprotective effect of the treatments with these agents.

Deficiency of M2 muscarinic acetylcholine receptors increases susceptibility of ventricular function to chronic adrenergic stress.

Suppressed parasympathetic nervous system (PSNS) function has been found in a variety of cardiovascular diseases, such as hypertension, heart failure, and diabetes. However, whether impaired PSNS function plays a significant role in ventricular dysfunction remains to be investigated. Cardiac regulation by the PSNS is primarily mediated by the M(2) muscarinic acetylcholine receptor (M(2)-AChR). In this study, we tested the hypothesis that lack of M(2)-AChR-mediated PSNS function may adversely impact cardiac ventricular function. Using M(2)-AChR knockout (KO) and wild-type (WT) mice, we found that the basal levels of heart rate and left ventricular function were similar in M(2)-AChR KO and WT mice. A bolus injection of isoproterenol (Iso) induced a greater increase in heart rate in M(2)-AChR KO mice than in WT mice. However, the responses of change in pressure over time (dP/dt) to Iso were similar in the two groups. After chronic infusion with Iso for 1 wk, the baseline values of left ventricular function were increased to similar extents in M(2)-AChR KO and WT mice. However, the M(2)-AChR KO mice exhibited impaired ventricular function, indicated as attenuated dP/dt and increased end-diastolic pressure, during an increase in cardiac afterload induced by a bolus injection of phenylephrine. Furthermore, chronic Iso infusion significantly increased matrix metalloproteinase (MMP) activity in the heart in M(2)-AChR KO mice. In primary culture of mixed neonatal rat cardiac fibroblast and cardiomyocytes, cotreatment with muscarinic agonist bethanechol reversed phenylephrine-induced increase in MMP-9 activation. These data suggest that M(2)-AChR may mediate an inhibitory regulation on MMP function. The overall results from this study suggest that M(2)-AChR-mediated PSNS function may provide cardiac protection. Lack of this protective mechanism will increase the susceptibility of the heart to cardiac stresses.

Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies.

Although heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic Escherichia coli (ETEC) have been documented as important factors associated with diarrheal diseases, investigations assessing the contributions of individual enterotoxins to the pathogenesis of E. coli infection have been limited. To address the individual roles of enterotoxins in the diarrheal disease caused by K88-positive ETEC in young pigs, enterotoxin-positive and -negative isogenic E. coli strains were constructed by using pBR322 to clone and express LT and STb. Four strains, K88+ astA, K88+ astA/pBR322, K88+ astA STb+, and K88+ astA LT+, were constructed and subsequently included in gnotobiotic piglet challenge studies, and their pathogenesis was assessed. The results indicated that all K88+ isogenic strains were able to colonize the small intestines of piglets exhibiting the K88 receptor. However, only LT- and STb-positive strains caused appreciable diarrhea. Piglets inoculated with the K88+ astA LT+ strain became dehydrated within 18 h, while those inoculated with the K88+ astA STb+ strain did not, although diarrhea developed in several piglets. The changes in the blood packed-cell volume and plasma total protein of gnotobiotic piglets inoculated with the LT-positive strains were significantly greater than those of pigs inoculated with the K88 astA/pBR322 strain (P = 0.012, P = 0.002). Immunochemistry image analysis also suggested that LT enhanced bacterial colonization in a gnotobiotic piglet model. This investigation suggested that LT is a major contributor to the virulence of K88+ ETEC and that isogenic constructs are a useful tool for studying the pathogenesis of ETEC infection.

Antibody repertoire development in fetal and neonatal piglets. IX. Three pathogen-associated molecular patterns act synergistically to allow germfree piglets to respond to type 2 thymus-independent and thymus-dependent antigens.

Newborn piglets maintained germfree (GF) cannot respond to either thymus-dependent (TD) or type 2 thymus-independent Ags (TI-2) unless colonized with bacteria. We show here that pathogen-associated molecular patterns (PAMPs), including muramyl dipeptide (MDP), LPS, and a B-class CpG oligonucleotide (CpG-B), can substitute for gut flora in the induction of neonatal immunoresponsiveness. These PAMPs alone or in combination had little effect on serum IgG and IgA levels, but CpG-B and CpG-B + MDP elevated total IgM levels 3- to 7-fold above that seen in colonized controls after booster immunization. Although only CpG-B could alone stimulate immunoresponsiveness, co-administration of LPS or MDP resulted in a 5-fold increase in the IgG response to both immunogens. Co-administered MDP did not promote secondary IgG responses to either Ag but instead pronounced secondary IgM responses to the epitopes of both immunogens. LPS co-administered with CpG-B may promote class switch recombination or cause differentiation of previously switched cells that become responsive after exposure to CpG-B. Primary and secondary IgG responses equally recognized the epitopes of the TI-2 and TD immunogens, whereas IgM responses favored the TI-2 epitope. Because PAMPs alone can result in Abs to 2,4,6-triitrophenyl and FLU without immunization, it suggests they alone cause differentiation of B cells of the preimmune repertoire. The finding that both bacterial PAMPs and colonization are capable of stimulating Ab responses in both immunized and nonimmunized piglets suggests that PAMPs derived from host flora may play a major role in awakening adaptive immunity in neonates.