Defying Gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and are a resource for understanding how plants regulate posture control. The peach (Prunus persica) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Little is known about the function of WEEP despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach trees do not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster lateral root gravitropic response. This suggests that WEEP moderates root gravitropism and is essential to establishing the set-point angle of lateral roots from the gravity vector. Additionally, size-exclusion chromatography indicated that WEEP proteins self-oligomerize, like other proteins with sterile alpha motif (SAM) domains. Collectively, our results from weeping peach provide insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Defying Gravity: WEEP promotes negative gravitropism in Prunus persica (peach) shoots and roots by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and serve as tremendous resources for understanding how plants regulate posture control. The Prunus persica (peach) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Until now, little was known about the function of WEEP protein despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach does not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster root gravitropic response, just as barley and wheat with mutations in their WEEP homolog EGT2. This suggests that the role of WEEP in regulating lateral organ angles and orientations during gravitropism may be conserved. Additionally, size-exclusion chromatography indicated that WEEP proteins self-oligomerize, like other SAM-domain proteins. This oligomerization may be required for WEEP to function in formation of protein complexes during auxin transport. Collectively, our results from weeping peach provide new insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Arabidopsis weep mutants exhibit narrow root angles.

Peach ( Prunus persica ) trees with a mutation in the weep gene exhibit a weeping branch phenotype. In contrast, Arabidopsis ( Arabidopsis thaliana ) weep mutants do not have a shoot architecture phenotype. A recent report revealed that barley ( Hordeum vulgare ) and wheat ( Triticum aestivum ) with mutations in EGT2, a WEEP homolog, have steeper root angles than standard varieties. We investigated the root architecture of three Arabidopsis weep mutant lines. All three lines exhibited steeper root angles and a smaller convex hull area, indicating that the total area explored by the root system is reduced. These results reveal WEEP is important for regulating lateral root angles in a dicot.

Oxygenation Performance of Different Non-Invasive Devices for Treatment of Decompression Illness and Carbon Monoxide Poisoning.

Study Objective: Application of high concentrations of oxygen to increase oxygen partial pressure (pO2) is the most important treatment for patients with carbon monoxide intoxication or divers with suspected decompression illness. The aim of this study was to evaluate the oxygenation performance of various non-invasive oxygen systems. Methods: The effect of different oxygen systems on arterial pO2, pCO2 and pH and their subjective comfort was evaluated in 30 healthy participants. Eight devices were included: nasal cannula, non-rebreather mask, AirLife Open mask, Flow-Safe II CPAP device, SuperNO2VA nasal PAP device, all operated with 15 L/min constant flow oxygen; nasal high-flow (50 L/min flow, 1.0 FiO2), non-invasive positive pressure ventilation (NPPV, 12 PEEP, 4 ASB, 1.0 FiO2) and a standard diving regulator (operated with pure oxygen). Results: Diving regulator, SuperNO2VA, nasal high-flow and NPPV achieved mean arterial pO2 concentrations between 538 and 556 mm Hg within 5 minutes. The AirLife Open mask, the nasal cannula and the non-rebreather mask achieved concentrations of 348-451 mm Hg and the Flow-Safe II device 270 mm Hg. Except for the AirLife open mask, pCO2 decreased and pH increased with all devices. The highest pH values were observed with NPPV, diving regulator, Flow-Safe II and nasal high-flow but apparent hyperventilation was uncommon. The AirLife Open and the non-rebreather mask were the most comfortable, the SuperNO2VA and the nasal cannula the most uncomfortable devices. Conclusion: A standard diving regulator and the SuperNO2VA device were equally effective in providing highest physiologically possible pO2 as compared to nasal high-flow and NPPV.

Livedo Racemosa - The Pathophysiology of Decompression-Associated Cutis Marmorata and Right/Left Shunt.

Decompression sickness and arterial gas embolism, collectively known as decompression illness (DCI), are serious medical conditions that can result from compressed gas diving. DCI can present with a wide range of physiologic and neurologic symptoms. In diving medicine, skin manifestations are usually described in general as cutis marmorata (CM). Mainly in the Anglo-American literature the terms cutis marmorata, livedo reticularis (LR), and livedo racemosa (LRC) are used interchangeably but actually describe pathophysiologically different phenomena. CM is a synonym for LR, which is a physiological and benign, livid circular discoloration with a net-like, symmetric, reversible, and uniform pattern. The decompression-associated skin discolorations, however, correspond to the pathological, irregular, broken netlike pattern of LRC. Unlike in diving medicine, in clinical medicine/dermatology the pathology of livedo racemosa is well described as a thrombotic/embolic occlusion of arteries. This concept of arterial occlusion suggests that the decompression-associated livedo racemosa may be also caused by arterial gas embolism. Recent studies have shown a high correlation of cardiac right/left (R/L) shunts with arterial gas embolism and skin bends in divers with unexplained DCI. To further investigate this hypothesis, a retrospective analysis was undertaken in a population of Austrian, Swiss, and German divers. The R/L shunt screening results of 18 divers who suffered from an unexplained decompression illness (DCI) and presented with livedo racemosa were retrospectively analyzed. All of the divers were diagnosed with a R/L shunt, 83% with a cardiac shunt [patent foramen ovale (PFO)/atrium septum defect (ASD)], and 17% with a non-cardiac shunt. We therefore not only confirm this hypothesis but when using appropriate echocardiographic techniques even found a 100% match between skin lesions and R/L shunt. In conclusion, in diving medicine the term cutis marmorata/livedo reticularis is used incorrectly for describing the actual pathology of livedo racemosa. Moreover, this pathology could be a good explanation for the high correlation of livedo racemosa with cardiac and non-cardiac right/left shunts in divers without omission of decompression procedures.

Carotid sinus syndrome: a case report of an unusual presentation of cardiac arrest while diving.

BACKGROUND: Carotid sinus syndrome (CSS) is an exaggerated response to carotid sinus baroreceptor stimulation, which may result in hypotension, prolonged asystole, and subsequently transient loss of consciousness due to cerebral hypoperfusion. However, this commonly benign syndrome may have lethal consequences under certain circumstances such as scuba diving. CASE SUMMARY: We report the case of a trained 73-year-old male diver, who survived an almost fatal diving accident without any neurological deficits due to cardiac arrest under water. After recovery and intensive diagnostics in the local hospital, the origin of cardiac arrest remained unclear. However, after referral to our tertiary care centre CSS could be diagnosed by provoking syncope and asystole with carotid sinus massage (CSM). Consequently, a leadless pacing system was implanted and his medical diving fitness could then be recertified. DISCUSSION: In conclusion, CSS may be an underdiagnosed cause of loss of consciousness. Thus, screening for CSS by CSM should be included in medical exams in senior athletes and specifically in senior divers.

The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu.

SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control.

A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling.

BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

Safety analysis of vagal nerve stimulation for continuous nerve monitoring during thyroid surgery.

OBJECTIVES/HYPOTHESIS: Intraoperative neuromonitoring (IONM) facilitates recurrent laryngeal nerve (RLN) identification, but various studies affirm virtually unchanged postoperative RLN palsy rates. Several authors meanwhile suggest continuous intraoperative neuromonitoring (CIONM) via vagal nerve stimulation (VNS) to improve RLN protection. However, knowledge of side effects of electrical VNS derives mainly from its therapeutic applications in the fields of neurology and psychiatry. The presented study was conducted to further evaluate the safety of CIONM and identify possible VNS related side effects. STUDY DESIGN: Prospective nonrandomized controlled trail. METHODS: Forty patients scheduled for thyroid or parathyroid surgery were enrolled in the trail. The intervention group consisted of 22 patients receiving VNS for CIONM. Eighteen patients were operated on with routine IONM. To assess VNS-induced effects on the autonomic nervous system (ANS), heart rate variability analysis (HRVA) was applied. Serum cytokine levels of tumor necrosis factor (TNF)-α were monitored to evaluate immunomodulatory effects of VNS. RESULTS: HRVA revealed significantly increased vagal activity during CIONM. This parasympathetic predominance was not countered by the sympathetic nervous system. Despite a significant increase of vagal tone, no hemodynamic events occurred; in fact, no significant changes in median heart rate or in median arterial blood pressure were detected. Even though anti-inflammatory effects of VNS have been reported, no attenuation of cytokine release of TNF-α was measured. CONCLUSIONS: VNS for CIONM resulted in increased vagal activity assessable via HRVA. The increased parasympathetic tone affected neither hemodynamics nor levels of the proinflammatory cytokine TNF-α. VNS for CIONM appears safe with the applied settings.

Protein phosphatase 2A (PP2A)-specific ubiquitin ligase MID1 is a sequence-dependent regulator of translation efficiency controlling 3-phosphoinositide-dependent protein kinase-1 (PDPK-1).

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells.

Impact of continuous intraoperative neuromonitoring on autonomic nervous system during thyroid surgery.

BACKGROUND: Continuous intraoperative neuromonitoring (CIONM) via vagal nerve stimulation (VNS) is a new option for recurrent laryngeal nerve (RLN) protection during thyroid surgery. The aim of this study was to evaluate the safety of VNS for CIONM and to assess its effects on the autonomic nervous system (ANS) through analyzing heart rate variability (HRV). METHODS: In a prospective, nonrandomized controlled study 5 patients received VNS for CIONM and 5 were operated on with conventional intermittent intraoperative neuromonitoring (IONM). HRV was analyzed in accord with patient-specific reference values. RESULTS: VNS resulted in significantly altered ANS balance. Relative parasympathetic activity increased during VNS. Yet, no relevant cardiac arrhythmias or hemodynamic alterations were observed during VNS. CONCLUSION: HRV analysis revealed a distinct impact of VNS for CIONM on ANS balance. VNS caused parasympathetic predominance that was not countered by increased sympathetic activity.

Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Point mutations in GLI3 lead to misregulation of its subcellular localization.

BACKGROUND: Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. PRINCIPAL FINDINGS: We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-alpha4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. CONCLUSIONS: The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations.

Phenotype variability in spinocerebellar ataxia type 2: a longitudinal family survey and a case featuring an unusual benign course of disease.

We report a 67 years old female patient out of a multigenerational family with spinocerebellar ataxia type 2 (SCA2) with an unusually benign course of disease. Although all SCA2 gene carriers have by now developed the predominant gait ataxia and brainstem oculomotor dysfunction, the index patient presented with a very mild course of disease, scoring only six points on the Scale for the Assessment and Rating of Ataxia after a disease duration of 13 years. Otherwise, intragenerational variability within family members such as the age at onset of disease and the course of disease was low. Reinvestigation of the genetic background variables in the SCA2 gene carrier reported here showed 27 repeats in the normal allele and 37 noninterrupted repeats in the abnormal allele. Interestingly, this patient has been taking lithium-carbonate over more than 30 years because of psychotic depression. Although anecdotic, this SCA2 case may provide promising insights into possible disease modifying mechanisms in SCA2.

A-overhang-dependent repeat expansion determination (ADRED).

In this study we present a quick and easy method for counting trinucleotide repeats by de-oxyadenosine overhang (A-overhang)-dependent repeat expansion determination (ADRED). During standard Taq DNA polymerase-based sequencing reactions, the unterminated sequencing products of short PCR fragments are tagged with a 3'-end A-overhang that is visible as an intense peak in an electropherogram; this allows for easy and precise determination of the fragment length and thus the extent of repeat expansions. ADRED has clear advantages over existing methods, because repeat numbers of both normal and pathogenic (expanded) alleles can be analyzed without using labeled primers or labeled DNA standards. Because ADRED includes a sequencing step, disease-relevant polymorphisms (e.g., CAA interruptions in spinocerebellar ataxia type 2) can simultaneously be detected.

The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex.

Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1alpha (EF-1alpha) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1alpha. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.

Immune responses to commercial equine vaccines against equine herpesvirus-1, equine influenza virus, eastern equine encephalomyelitis, and tetanus.

Horses are commonly vaccinated to protect against pathogens which are responsible for diseases which are endemic within the general horse population, such as equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1), and against a variety of diseases which are less common but which lead to greater morbidity and mortality, such as eastern equine encephalomyelitis virus (EEE) and tetanus. This study consisted of two trials which investigated the antigenicity of commercially available vaccines licensed in the USA to protect against EIV, EHV-1 respiratory disease, EHV-1 abortion, EEE and tetanus in horses. Trial I was conducted to compare serological responses to vaccines produced by three manufacturers against EIV, EHV-1 (respiratory disease), EEE, and tetanus given as multivalent preparations or as multiple vaccine courses. Trial II compared vaccines from two manufacturers licensed to protect against EHV-1 abortion, and measured EHV-1-specific interferon-gamma (IFN-gamma) mRNA production in addition to serological evidence of antigenicity. In Trial I significant differences were found between the antigenicity of different commercial vaccines that should be considered in product selection. It was difficult to identify vaccines that generate significant immune responses to respiratory viruses. The most dramatic differences in vaccine performance occurred in the case of the tetanus antigen. In Trial II both vaccines generated significant antibody responses and showed evidence of EHV-1-specific IFN-gamma mRNA responses. Overall there were wide variations in vaccine response, and the vaccines with the best responses were not produced by a single manufacturer. Differences in vaccine performance may have resulted from differences in antigen load and adjuvant formulation.

Cytokine responses to EHV-1 infection in immune and non-immune ponies.

Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups were subjected to an intranasal challenge infection with EHV-1, and blood samples were collected pre-infection, and at 7 and 21 days post-infection for preparation of PBMCs. At each time point, the in vitro responses of PBMCs to stimulation with EHV-1 were measured, including IFN-gamma and IL-4 mRNA production, and lymphoproliferation. Group 1 ponies showed no signs of clinical disease or viral shedding after challenge infection. Group 2 ponies experienced a biphasic pyrexia, mucopurulent nasal discharge, and nasal shedding of virus after infection. Group 1 ponies had an immune response characterized both before and subsequent to challenge infection by an IFN-gamma response to EHV-1 in the absence of an IL-4 response, and demonstrated increased EHV-1-specific lymphoproliferation post-infection. Group 2 ponies had limited cytokine or lymphoproliferative responses to EHV-1 pre-challenge, and demonstrated increases in both IFN-gamma and IL-4 responses post-challenge, but without any lymphoproliferative response. Protective immunity to EHV-1 infection was therefore characterized by a polarized IFN-gamma dependent immunoregulatory cytokine response.

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.