Rabbit Polyclonal Antiserum Research Articles

The Size and Conservation of a Coiled-coil Structure in the Ectodomain of Human BST-2/Tetherin Is Dispensable for Inhibition of HIV-1 Virion Release

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.

Studies on the O-antigen of Hafnia alvei PCM 1224 structurally and serologically related to the O-antigen of H. alvei 481-L

Mild hydrolysis of the lipopolysaccharide of Hafnia alvei PCM 1224 at pH 4.2 cleaved partially the O-polysaccharide chain by the glycosyl phosphate linkages to yield a phosphorylated hexasaccharide representing the repeating unit of the O-polysaccharide and higher oligosaccharides consisting of two or more repeating units. Studies of the degradation products before and after dephosphorylation by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HSQC-TOCSY, and 1H,31P HMBC experiments, enabled elucidation of the following structure of the O-polysaccharide: [Display omitted] This structure is similar to that of the O-polysaccharide of H. alvei 481-L established earlier (Kubler-Kielb, J.; Vinogradov, E.; Garcia Fernandez, J. M.; Szostko, B.; Zwiefka, A.; Gamian, A. Carbohydr. Res.2006, 341, 2980–2985), which has the same sugar composition and the same main chain structure and differs in the site of attachment of α-d-Glcp only. A two-way serological cross-reactivity of the lipopolysaccharides of H. alvei PCM 1224 and 481-L with polyclonal rabbit antisera indicated the expediency of classification of both strains to the same O-serogroup.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

Specific Residues of a Conserved Domain in the N Terminus of the Human Cytomegalovirus pUL50 Protein Determine Its Intranuclear Interaction with pUL53

Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3-20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.

Unexpected Immunostaining of Mycobaterium leprae With a Polyclonal Antibody Against Treponema pallidum

Unexpected Immunostaining of Mycobaterium leprae With a Polyclonal Antibody Against Treponema pallidum

In Vitro Expression and Production of Antibody Against Cymbidium mosaic virus Coat Protein

Polyclonal rabbit antisera were produced using coat protein of Cymbidium mosaic virus (CymMV) Indian isolate expressed in E. coli as GST fusion. The expressed protein was purified by GST-fusion protein purification kit for use as an immunogen in rabbits. Antisera prepared in this manner reacted in double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) with extract from CymMV-infected tissue. The results indicate that polyclonal antisera prepared from expressed CymMV coat proteins were useful for the detection of CymMV in an array of assays. The detection system developed is highly effective for detection of Indian strain of the virus in comparison to kits available in the international market.

Plasma myostatin measured by a competitive ELISA using a highly specific antiserum

BackgroundUnderstanding the (patho)physiology of the negative muscle regulator myostatin (Myo) is important for patients with skeletal muscle disorders or cardiac disease. However, a reliable tool for measuring plasma Myo immunoreactivity is still lacking. MethodsHuman full-length proMyo was used to raise a polyclonal rabbit antiserum for a competitive Myo ELISA that was validated in patients with decompensated congestive heart failure (CHF) and in control patients (n=20 each). ResultsThe Myo antiserum detected all subunits of human proMyo. The calibration curve showed an optimal range between 0.3 and 83.3ng/ml (7.5–2100pmol/l), with no cross-reactivity to growth differentiation factor-11, follistatin and follistatin-related gene protein. The inter-assay and intra-assay variances in human serum were ≤15% and ≤10%, respectively; the detection limit was 270pg/ml (6.75pmol/l). The assay showed excellent linearity in human plasma. Plasma NT-proBNP and Myo were significantly elevated in decompensated CHF compared with control patients and decreased significantly upon recompensating therapy. ConclusionWe describe the development of the first ELISA for myostatin immunoreactivity and its validation during recompensating therapy for CHF. This assay will be valuable for investigating neurological and cardiac diseases and states of cachexia, insulin resistance, and obesity.

Abstract 1258: High-density protein lysate arrays: an essential tool in assessing antibody specificity for clinical applications

Abstract Antibodies for clinical applications require rigorous scrutiny qualifying them for diagnostics or therapeutic use. However, this is a lengthily and complicated process that provides only limited conformation. Protein microarrays are new tools that can determine the specificity and reactivity of Abs and help identifying their target in less than a day. We created a high-density protein array comprised from more than 10,000 over-expressed protein lysates made in human HEK293T cells. We tested more than 15 available antibodies to two cancer targets; the excision repair cross-complementation group 1 (ERCC1) and the receptor tyrosine kinase ERBB2 (Her2/neo). Our results demonstrated the ability of the lysate array in determining the specificity of antibodies with a single fast and easy assay. ERCC1 is a predictor of resistance to platinum based drugs and for poor prognosis in cancer. The commonly used anti ERCC1 antibody, mAb 8F1, is known to give non-specific cytoplasmic staining and to cross-react with an unknown protein of similar size. We tested 10 commercially available Abs to ERCC1 and identified four mono-specific ones (TrueMAb OriGene). Most of these specific Abs gave higher reactivity than clone 8F1. With this test we also identified a cytoplasmic protein of appropriate molecular weight that is cross-reacting with mAb 8F1. The above results were corroborated by WB analysis and IHC assays supporting the validity of this type of assays. We extended our tests to include anti ERBB2 Abs as this important biomarker is indicative for the use and response to the drug Herceptin. Our tests included mono and poly clonal antibodies and clone 4B5, a rabbit mAb currently used for IHC diagnostic. We found clone 4B5 to be reactive with two additional proteins besides ERBB2, one of them is ERBB4. We also tested a rabbit polyclonal anti ERBB2 serum and found it to be reactive with additional proteins unrelated to those recognized by mAb 4B5. Three out of the four mouse mAbs tested (TrueMAb OriGene) were determined to be specific. We submit that the over-expression protein lysate arrays can establish the reactivity and specificity of Abs and identify unknown targets. The speed and convenience of these assays make them an important tool in developing Abs for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1258. doi:1538-7445.AM2012-1258

Immunohistochemical detection of piscine reovirus (PRV) in hearts of Atlantic salmon coincide with the course of heart and skeletal muscle inflammation (HSMI)

Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins μ1C and σ1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes.

Size and Conformation Limits to Secretion of Disulfide-bonded Loops in Autotransporter Proteins

Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

C-terminal Hydrophobic Region in Human Bone Marrow Stromal Cell Antigen 2 (BST-2)/Tetherin Protein Functions as Second Transmembrane Motif

BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.

Eggplant polyphenol oxidase multigene family: Cloning, phylogeny, expression analyses and immunolocalization in response to wounding

Though polyphenol oxidase (PPO) genes from tomato and potato have been extensively studied, information about PPO genes in eggplant ( Solanum melongena) is lacking. The main objective of this study is to understand the structural and functional aspects of eggplant PPO genes. Six eggplant PPO genes ( SmePPO1–6) cloned by RACE and genome walking were found to be intronless and correspond to eight eggplant unigenes. Comprehensive sequence analyses indicated that the eggplant PPO genes exhibit considerable variation in the transit peptide regions, copper-binding domains and UTRs, and fall into two distinct structural classes. Further, PPO gene members appear to exist in clusters on eggplant chromosome 8 as seen in the case of tomato and potato PPOs. During normal growth and development, SmePPO1 and 2 are expressed in roots, whereas the transcript levels of all the eggplant PPO genes vary considerably in leaves, flowers and fruits. SmePPO1 was expressed in Escherichia coli as a GST fusion protein, and immunoblot using rabbit polyclonal antiserum to GST–SmePPO1 detected a major protein band (∼70 kDa) and a minor band (∼67 kDa) in eggplant fruit extract. Tissue printing indicated the predominant presence of PPO in the exocarp and the areas surrounding the seeds in the mesocarp of eggplant fruits. Immunolocalization of PPOs in eggplant infested with shoot-and-fruit borer revealed localization of the PPO at the site of infection in tender shoots and fruits, and further inside the mature tissues. The upregulation of eggplant PPO gene transcripts following mechanical injury shows that all the genes except SmePPO2 are induced in the fruit over 6 h. On the contrary, the transcripts of SmePPO2 and PPO3 are not detectable in the stem, and expression seems to be prominent over a 2 h period for SmePPO1 and SmePPO4–6. Our results show that eggplant PPO genes are structurally different, and are differentially expressed in various tissues of eggplant indicating their functional diversity.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t)SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e.g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EH rats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection

The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell receptors and therefore specific cell types. It is also an important immune target for the host in neutralizing the virus. Four antigenic sites A, B, C, and D that reside near the N-terminal domain have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. Herein, we enzymatically amplified, cloned, and expressed the S-AD fragment from TGEV in the prokaryotic expression vector, pET-30a. Maximum protein expression was achieved at 30°C over a 5-h period post-induction. Rabbit polyclonal antiserum was generated using recombinant S-AD (rS-AD) protein. In contrast to prior studies showing no activity with bacterially produced S protein, results indicated that polyclonal serum recognized TGEV-infected cells and reduced infection by 100%. Furthermore, the truncated rS-AD peptide was able to bind to the surface of cells from swine testes in a competitive manner and completely inhibit viral infection.

Human Lysophosphatidylcholine Acyltransferases 1 and 2 Are Located in Lipid Droplets Where They Catalyze the Formation of Phosphatidylcholine

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.

Pathology and immunohistochemistry of papillomavirus-associated cutaneous lesions in Cape mountain zebra, giraffe, sable antelope and African buffalo in South Africa

Skin lesions associated with papillomaviruses have been reported in many animal species and man. Bovine papillomavirus (BVP) affects mainly the epidermis, but also the dermis in several species including bovine, the best-known example being equine sarcoid, which is associated with BVP types 1 and 2. This publication describes and illustrates the macroscopic and histological appearance of BPV-associated papillomatous, fibropapillomatous or sarcoid-like lesions in Cape mountain zebra (Equus zebra zebra) from the Gariep Dam Nature Reserve, 2 giraffes (Giraffa camelopardalis) from the Kruger National Park, and a sable antelope (Hippotragus niger) from the Kimberley area of South Africa. An African buffalo (Syncerus caffer) cow from Kruger National Park also had papillomatous lesions but molecular characterisation of lesional virus was not done. Immunohistochemical staining using polyclonal rabbit antiserum to chemically disrupted BPV-1, which cross-reacts with the L1 capsid of most known papillomaviruses, was positive in cells of the stratum granulosum of lesions in Giraffe 1, the sable and the buffalo and negative in those of the zebra and Giraffe 2. Fibropapillomatous and sarcoid-like lesions from an adult bovine were used as positive control for the immunohistochemistry and are described and the immunohistochemistry illustrated for comparison. Macroscopically, both adult female giraffe had severely thickened multifocal to coalescing nodular and occasionally ulcerated lesions of the head, neck and trunk with local poorly-circumscribed invasion into the subcutis. Necropsy performed on the 2nd giraffe revealed neither internal metastases nor serious underlying disease. Giraffe 1 had scattered, and Giraffe 2 numerous, large, anaplastic, at times indistinctly multinucleated dermal fibroblasts with bizarre nuclei within the sarcoid-like lesions, which were BPV-1 positive in Giraffe 1 and BPV-1 and -2 positive in Giraffe 2 by RT-PCR. The sable antelope presented with a solitary large lesion just proximal to the right hind hoof, which recurred after excision, and was BPV-1 positive by RT-PCR. Other wart-like growths were present elsewhere on the body. The Cape mountain zebra either succumbed from their massive lesions or were euthanased or removed from the herd because of them. The lesions wereBPV-1 and/or -2 positive byRT-PCR. The buffalo lesions were wart-like papillomatous projections in the inguinal and udder region. Stratum granulosum cells that stained immunohistochemically positive in the various species appeared koilocyte-like, as described in human papillomaviral lesions.

Pathology and immunohistochemistry of papillomavirus-associated cutaneous lesions in Cape mountain zebra, giraffe, sable antelope and African buffalo in South Africa

Skin lesions associated with papillomaviruses have been reported in many animal species and man. Bovine papillomavirus (BVP) affects mainly the epidermis, but also the dermis in several species including bovine, the best-known example being equine sarcoid, which is associated with BVP types 1 and 2. This publication describes and illustrates the macroscopic and histological appearance of BPV-associated papillomatous, fibropapillomatous or sarcoid-like lesions in Cape mountain zebra (Equus zebra zebra) from the Gariep Dam Nature Reserve, 2 giraffes (Giraffa camelopardalis) from the Kruger National Park, and a sable antelope (Hippotragus niger) from the Kimberley area of South Africa. An African buffalo (Syncerus caffer) cow from Kruger National Park also had papillomatous lesions but molecular characterisation of lesional virus was not done. Immunohistochemical staining using polyclonal rabbit antiserum to chemically disrupted BPV-1, which cross-reacts with the L1 capsid of most known papillomaviruses, was positive in cells of the stratum granulosum of lesions in Giraffe 1, the sable and the buffalo and negative in those of the zebra and Giraffe 2. Fibropapillomatous and sarcoid-like lesions from an adult bovine were used as positive control for the immunohistochemistry and are described and the immunohistochemistry illustrated for comparison. Macroscopically, both adult female giraffe had severely thickened multifocal to coalescing nodular and occasionally ulcerated lesions of the head, neck and trunk with local poorly-circumscribed invasion into the subcutis. Necropsy performed on the 2nd giraffe revealed neither internal metastases nor serious underlying disease. Giraffe 1 had scattered, and Giraffe 2 numerous, large, anaplastic, at times indistinctly multinucleated dermal fibroblasts with bizarre nuclei within the sarcoid-like lesions, which were BPV-1 positive in Giraffe 1 and BPV-1 and -2 positive in Giraffe 2 by RT-PCR. The sable antelope presented with a solitary large lesion just proximal to the right hind hoof, which recurred after excision, and was BPV-1 positive by RT-PCR. Other wart-like growths were present elsewhere on the body. The Cape mountain zebra either succumbed from their massive lesions or were euthanased or removed from the herd because of them. The lesions were BPV-1 and/or -2 positive by RT-PCR. The buffalo lesions were wart-like papillomatous projections in the inguinal and udder region. Stratum granulosum cells that stained immunohistochemically positive in the various species appeared koilocyte-like, as described in human papillomaviral lesions.

Identification of Key Amino Acid Residues That Determine the Ability of High Risk HPV16-E7 to Dysregulate Major Histocompatibility Complex Class I Expression

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

Immunoperoxidase inhibition assay for rabies antibody detection

An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG–peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the “simplified fluorescence inhibition microtest” (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available.

Overexpression of Glutaminyl Cyclase, the Enzyme Responsible for Pyroglutamate Aβ Formation, Induces Behavioral Deficits, and Glutaminyl Cyclase Knock-out Rescues the Behavioral Phenotype in 5XFAD Mice

Pyroglutamate-modified Aβ (AβpE3-42) peptides are gaining considerable attention as potential key players in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity. Overexpressing AβpE3-42 induced a severe neuron loss and neurological phenotype in TBA2 mice. In vitro and in vivo experiments have recently proven that the enzyme glutaminyl cyclase (QC) catalyzes the formation of AβpE3-42. The aim of the present work was to analyze the role of QC in an AD mouse model with abundant AβpE3-42 formation. 5XFAD mice were crossed with transgenic mice expressing human QC (hQC) under the control of the Thy1 promoter. 5XFAD/hQC bigenic mice showed significant elevation in TBS, SDS, and formic acid-soluble AβpE3-42 peptides and aggregation in plaques. In 6-month-old 5XFAD/hQC mice, a significant motor and working memory impairment developed compared with 5XFAD. The contribution of endogenous QC was studied by generating 5XFAD/QC-KO mice (mouse QC knock-out). 5XFAD/QC-KO mice showed a significant rescue of the wild-type mice behavioral phenotype, demonstrating the important contribution of endogenous mouse QC and transgenic overexpressed QC. These data clearly demonstrate that QC is crucial for modulating AβpE3-42 levels in vivo and prove on a genetic base the concept that reduction of QC activity is a promising new therapeutic approach for AD.