anti-H Antibodies Research Articles

Hydrogel based QCM aptasensor for detection of avian influenzavirus

The objective of this study was to develop a quartz crystal microbalance (QCM) aptasensor based on ssDNA crosslinked polymeric hydrogel for rapid, sensitive and specific detection of avian influenza virus (AIV) H5N1. A selected aptamer with high affinity and specificity against AIV H5N1 surface protein was used, and hybridization between the aptamer and ssDNA formed the crosslinker in the polymer hydrogel. The aptamer hydrogel was immobilized on the gold surface of QCM sensor using a self-assembled monolayer method. The hydrogel remained in the state of shrink if no H5N1 virus was present in the sample because of the crosslinking between the aptamer and ssDNA in the polymer network. When it exposed to target virus, the binding reaction between the aptamer and H5N1 virus caused the dissolution of the linkage between the aptamer and ssDNA, resulting in the abrupt swelling of the hydrogel. The swollen hydrogel was monitored by the QCM sensor in terms of decreased frequency. Three polymeric hydrogels with different ratio (100:1 hydrogel I, 10:1 hydrogel II, 1:1 hydrogel III) of acrylamide and the aptamer monomer were synthesized, respectively, and then were used as the QCM sensor coating material. The results showed that the developed hydrogel QCM aptasensor was capable of detecting target H5N1 virus, and among the three developed aptamer hydrogels, hydrogel III coated QCM aptasensor achieved the highest sensitivity with the detection limit of 0.0128HAU (HA unit). The total detection time from sampling to detection was only 30min. In comparison with the anti-H5 antibody coated QCM immunosensor, the hydrogel QCM aptasensor lowered the detection limit and reduced the detectiontime.

Significance of serological monitoring in a Bombay Rh (D) negative phenotype pregnant woman: A case report

A 32year old Indian female was referred to our hospital at 32weeks of gestation because of difficulty in blood group determination and further antenatal care. The results of cell and serum grouping of her blood sample were suggestive of Bombay (Oh) Rh (D) negative phenotype. An indirect antiglobulin test (IAT) using a pool of red cells from two Bombay Rh (D) positive blood donors gave negative result using the tube as well as the gel technique (LISS-Coombs Card, BioRad, Switzerland), thus ruling out anti-D antibody in her serum. The anti-H titer was 16 (tube technique) and with dithiothreitol (DTT) treated patient’s serum the antibody screening was negative suggestive of IgM type of anti-H antibodies. Within the patient’s family, only one member (younger sister) was of Oh phenotype and also was Rh (D) negative. The baby was born vaginally at 38+6weeks of gestation and was non-hydropic with a packed cell volume (PCV) of 55%. The baby’s blood group was AB Rh (D) negative and the cord blood direct antiglobulin test (DAT) was negative. Thus, a careful serological testing of Oh phenotype antenatal women especially with Rh (D) negative phenotype is of utmost importance in determining the isoimmunization status.

Bombay Phenotype: Report of 2 Cases

Two cases of the rare blood group “Bombay phenotype” are discussed here. This rare blood group, Bombay (Oh) was first established by Bhende et al in Bombay (Mumbai), in 1952.In the ABO (ABH) blood group system, the ‘O’ antigen represents the lack of A or B antigens; however it has the most amount of H antigen. If the H gene is absent, which is extremely rare, H substance can not be formed and subsequent A and B antigens can not also be formed. Absence of H gene results in the Bombay phenotype (Oh) 1-6, 7-13. Individuals with the Bombay phenotype develop anti-H antibodies. This is the reason that undetected Bombay individuals (typically typed as O individuals) will be cross match incompatible with O individuals. Bombay phenotype individuals can only receive blood from other Bombay individuals. In India, 1 in 10,000 has been found to have “Bombay” blood group. Where as, in Bangladesh till now only nine persons with Bombay (Oh) blood group have been formally reported by the Transfusion Medicine Department of Bangabandhu Sheikh Mujib Medical University (BSMMU). Using two cases we would like to discuss strategies to properly diagnose cases of Bombay phenotype in Bangladesh DOI: http://dx.doi.org/10.3329/jbcps.v29i4.11347 J Bangladesh Coll Phys Surg 2011; 29: 241-243

Seroprevalence of subtype H3 influenza A virus in South Korean cats.

To investigate the potential transmission of subtype H3 influenza virus to cats, a serological survey was carried out in South Korea. Serum samples (n=1027) were obtained from 809 pet cats and 218 domesticated cats living in urban colonies (D-cats) from 2008 to 2010, and tested using an influenza anti-nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and the haemagglutination inhibition (HI) test, which was recommended by the World Organization for Animal Health. Anti-influenza virus antibodies were detected in 3.12% and 2.43% of cat sera tested using the NP-specific ELISA and HI test, respectively. Anti-H3 antibodies were also identified when the HI assay was used for influenza virus serotyping. These data may indicate the sporadic transmission of subtype H3 influenza virus from other infected species to cats in South Korea.

The standard mouse assay of anti-venom quality does not measure antibodies neutralising the haemorrhagic activity of Vipera ammodytes venom

The venom of Vipera ammodytes ammodytes (Vaa), like the venoms of other Viperinae snakes, is largely haemorrhagic and necrotising, and only to a lesser extent neurotoxic to humans. The components most extensively studied so far, and most probably involved in generating the observed pathologies, are haemorrhagins (H), members of the metalloproteinase group of enzymes, and neurotoxic ammodytoxins (Atxs), that belong to the secretory phospholipases A2. Rabbit antisera were prepared containing functional antibodies specific for each class of pathology-inducing venom constituents and for both classes together. The involvement of these antibodies in neutralising the toxicity of whole Vaa venom was assessed using the ED50 assay in mice. This assay is the only regulatorily approved assay for estimating anti-venom potency and as such has the task to quantify the active compound neutralising venom-induced pathology of the anti-venom. Fully functional anti-Atx antibodies were shown to be responsible for neutralising the portion of venom toxicity, while anti-H antibodies were not protective in this assay. Thus, the mouse ED50 assay, intended to measure the active principle of the anti-venom, does not measure antibodies specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view.

A nanoforest structure for practical surface-enhanced Raman scattering substrates

A nanoforest structure for surface-enhanced Raman scattering (SERS) active substrates is fabricated and analyzed. The detailed morphology of the resulting structure can be easily controlled by modifying the process parameters such as initial gold layer thickness and etching time. The applicability of the nanoforest substrate as a label-free SERS immunosensor is demonstrated using influenza A virus subtype H1N1. Selective binding of the H1N1 surface antigen and the anti-H1 antibody is directly detected by the SERS signal differences. Simple fabrication and high throughput with strong in-plane hot-spots imply that the nanoforest structure can be a practical sensing component of a chip-based SERS sensing system.

Seroprevalence of avian origin H3N2 canine influenza virus infection in pet dogs in Shenzhen, China

Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory disease in dogs. Canine influenza is caused by two subtypes of influenza. A virus: H3N2 and H3N8. In recent years, surveys of avian origin CIV infection in dogs have been reported worldwide. However, little is known about the prevalence of CIV in pet dogs in China. In the present study, the prevalence of avian origin CIV H3N2 in pet dogs in Shenzhen, Southern China was investigated using the enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) assay.Thirty-one (6.71%) of the 462 serum samples tested were seropositive for avian origin CIV by ELISA. Use of the HI test revealed the presence of anti-H3 antibodies in 28 (6.06%) of 462 serum samples. The prevalence ranged from 4.87% (HI) or 6.19% (ELISA) to 7.41% among dogs of different ages, with high prevalence in pet dogs of 1 to -3 years old, but low prevalence in pet dogs≤1 year. The seroprevalence in female dogs was 5.21%, and in male dogs it was 7.78% (ELISA) or 6.67% (HI). These findings demonstrated that avian origin canine influenza virus infection is prevalent in pet dogs and can spread rapidly through local dog populations, which indicates its potential for becoming established in pet dogs throughout China. Key words: Canine influenza virus, seropervalence, pet dog, enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assay.

A Two-in-One Antibody against HER3 and EGFR Has Superior Inhibitory Activity Compared with Monospecific Antibodies

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling invitro and invivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30 nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2 h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody–antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.

Bombay Blood Type and Use of Red Cell Apheresis During Pregnancy

Bombay blood type is among the rarest in the world and is associated with anti-H antibodies. These antibodies cause severe hemolysis when exposed to any blood product except Bombay and have been reported to cause hemolytic disease of the newborn. A primigravid woman with Bombay blood type received erythropoietin and underwent autologous blood donation using red cell apheresis. Maternal anti-H immunoglobulin G antibodies were identified and serial evaluation of the middle cerebral artery peak systolic velocity was used to monitor for fetal anemia. Hemolytic disease of the newborn did not develop. Red cell apheresis is a valuable procedure that results in two units of red blood cells per donation and was well tolerated during pregnancy.

Bovine Antibody-Based Oral Immunotherapy for Reduction of Intragastric Helicobacter pylori Colonization: A Randomized Clinical Trial

Antibiotic-based regimens are frequently used for the treatment of Helicobacter pylori infection. These regimens fail to eradicate H pylori in 15% to 40% of patients, primarily due to antimicrobial resistance and insufficient patient compliance. Effective prevention and eradication of H pylori by passive immunization with orally administered bovine antibodies has been demonstrated in animal studies, and may serve as an alternative therapy in humans. To study the efficacy and safety of orally administered bovine anti-H pylori antibodies for the reduction of intragastric bacterial load and eradication of H pylori in humans. Dairy cows were immunized against H pylori. After confirmation of the presence of anti-H pylori antibodies in the milk, the milk was subsequently processed into a whey protein concentrate (WPC). In a prospective, double-blind, placebo-controlled randomized clinical trial, H pylori-infected subjects were randomly assigned to treatment with the WPC preparation or placebo. Study medication was continued for 28 days; subjects were followed-up for 56 days. Of the 30 subjects included, 27 completed the protocol. Of these 27 evaluable subjects, 14 were treated with WPC and 13 with placebo. There was no significant difference in urea breath test decrease between the WPC- and placebo-treated group (P=0.75). H pylori-associated gastritis and density were not significantly reduced in either group after treatment (P>0.05 for all). Bovine antibody-based oral immunotherapy appears to be safe, but does not significantly reduce intragastric density in humans. Further studies are needed to determine whether WPC treatment has additional value to conventional antibiotic treatment for H pylori.

Fatal cold agglutinin-induced haemolytic anaemia: a case report

IntroductionCold agglutinin disease usually develops as a result of the production of a specific immunoglobulin M auto-antibody directed against the I/i and H antigens, precursors of the ABH and Lewis blood group substances, on red blood cells. Autoimmune and lymphoproliferative disorders, Mycoplasma pneumoniae and other infections can be associated with the production of cold agglutinins. In its classic presentation with haemolytic anaemia and Raynaud's syndrome, cold agglutinin disease is usually idiopathic. Several factors play a role in determining the ability of a cold agglutinin to induce a haemolytic anaemia such as antibody concentration and temperature range, in particular the highest temperature at which antibodies interact with red blood cells.Case presentationA 48-year-old Caucasian man presented to our hospital with symptoms of extreme asthenia caused by severe anaemia. The transfusion of red blood cells (O Rh-positive), started as prescribed by the emergency guidelines in force without pre-transfusion tests, induced fatal haemolysis because of the presence of high levels of anti-H antibodies in his blood, that reacted with the large amount of H antigen in universal (0) red blood cells.ConclusionEmergency transfusion of universal red blood cells (0 Rh-positive or negative) is usually accepted by the international guidelines in force in emergency departments. In this report we describe a rare complication caused by the very high concentration in the recipient of cold agglutinins and the activation of the complement system, responsible for red blood cell lysis and consequent fatal cardiovascular shock. We conclude that emergency transfusion of universal red blood cells (0 Rh-positive or negative) may be dangerous and its risk should be assessed against the risk of delaying transfusion until the pre-transfusion tests are completed.

Avian influenza H5N1 virus infections in vaccinated commercial and backyard poultry in Egypt

In this paper, we describe results from a high-pathogenic H5N1 avian influenza virus (AIV) surveillance program in previously H5-vaccinated commercial and family-backyard poultry flocks that was conducted from 2007 to 2008 by the Egyptian National Laboratory for Veterinary Quality Control on Poultry Production. The real-time reverse transcription PCR assay was used to detect the influenza A virus matrix gene and detection of the H5 and N1 subtypes was accomplished using a commercially available kit real-time reverse transcription PCR assay. The virus was detected in 35/3,610 (0.97%) and 27/8,682 (0.31%) of examined commercial poultry farms and 246/816 (30%) and 89/1,723 (5.2%) of backyard flocks in 2007 and 2008, respectively. Positive flocks were identified throughout the year, with the highest frequencies occurring during the winter months. Anti-H5 serum antibody titers in selected commercial poultry ranged from <2 (negative) to 9.6 log(2) when determined in the hemagglutination inhibition test using a H5 AIV antigen. In conclusion, despite the nationwide vaccination strategy of poultry in Egypt to combat H5N1 AIV, continuous circulation of the virus in vaccinated commercial and backyard poultry was reported and the efficacy of the vaccination using a challenge model with the current circulating field virus should be revised.

A generic surface chemistry for peptide microarrays implementation: Application to the detection of anti-H3 antibody

Peptide microarray can be implemented by immobilization of peptides on a solid support or by direct on-chip peptide synthesis (OCPS). In the first case, peptide primary sequences can be ensured prior to their immobilization but structural diversity is achieved at high cost in terms of reagents. In the second case, high diversity is achieved with low amount of reagents but the primary and secondary structures cannot be ascertained. In both cases, the immobilization step will influence the overall biological activity. We proposed a strategy where direct peptide on-chip synthesis and peptide immobilization are viewed as complementary approaches. In a first step, OCPS is envisioned for the screening and selection of biologically relevant peptides. In a second step, selected peptides would be synthesized on resin, qualified and immobilized for implementing microarrays (i.e. for diagnosis). A versatile surface chemistry for both OCPS and peptide immobilization was developed allowing for an identical physico-chemical environment for both implementation strategies. In the present report, a 16 mer peptide corresponding to the human histone H3 epitope was synthesized on an amino-functionalized support. Surface stability (including upon deprotection steps) and peptide primary and secondary structures were assessed with Cy3–streptavidine conjugates and with immunonassays. Peptides, either on-chip synthesized or immobilized, exhibited a similar biological activity.

Immunomagnetic separation of Vibrio vulnificus with antiflagellar monoclonal antibody.

Raw oysters are primary vectors for Vibrio vulnificus infections, and a rapid detection method for V. vulnificus in raw oysters before distribution would be an indispensable tool for the seafood industry. One approach to improving the recovery and detection of V. vulnificus without sacrificing assay time is through the use of immunomagnetic separation (IMS). The aim of this study was to develop and optimize an IMS protocol using anti-H (antiflagellar) antibody for determining the level of V. vulnificus in phosphate-buffered saline (PBS) suspensions and spiked oyster homogenate. Six monoclonal antibodies were produced by immunizing mice at 2-week intervals by injection of 50 microg of purified V. vulnificus ATCC 27562 flagellin. Antibodies that exhibited high anti-H titers were coated onto Cowan I Staphylococcus aureus cells and sheep anti-mouse immunoglobulin G immunomagnetic beads. The two reagents were used to determine the species specificity of the selected antibodies, which positively identified and coagglutinated 70 isolates identified genetically as V. vulnificus and did not react with 40 Vibrio parahaemolyticus isolates or nine other Vibrio species. The IMS protocol was optimized for PBS and oyster homogenate spiked with three different strains of V. vulnificus. IMS with V. vulnificus-spiked PBS yielded binding of 19 to 57%, and IMS with spiked oyster homogenate carried out at two V. vulnificus levels exhibited binding of 25 to 57%. The IMS protocol for V. vulnificus could be used to concentrate and detect V. vulnificus in seawater and shellfish homogenate.

Kinetics of neutralizing antibodies in patients naturally infected by H5N1 virus.

BackgroundLittle is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection.MethodsUsing H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1–139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1–2 month –11 months after exposure).ResultsOf 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre ≥80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1–2 months and 173 at 10–12 months and thus were higher than the titers from asymptomatic individuals (149 at 1–2 months, 62.2 at 10–12 months). Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10–11 months after exposure in asymptomatically infected individuals tested negative.ConclusionsOur results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies.

Establishment of heparan sulphate deficient primary endothelial cells from EXT-1flox/flox mouse lungs and sprouting aortas

Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1(flox/flox) mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.

Simultaneous over activation of EGFR, telomerase (h TERT), and cyclin D1 correlates with advanced disease in larynx squamous cell carcinoma: a tissue microarray analysis

Overexpression of Epidermal Growth Factor Receptor (EGFR) and also of cell cycle control proteins, such as cyclin D1 is a frequent event in squamous cell carcinoma of the larynx (LSSC). Our aim was to correlate their protein levels with telomerase catalytic subunit (h-TERT) expression. Using tissue microarray technology, fifty-five paraffin embedded histologically confirmed primary LSSCs and also ten dysplastic lesions were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) was performed by the use of anti-EGFR, anti-cyclin D1, and anti-h TERT monoclonal antibodies. Chromogenic in situ hybridization (CISH) analysis was also applied using EGFR gene and chromosome 7 probes, respectively. EGFR, cyclin D1 and h-TERT protein overexpression was observed in 48/55 (87.2%), 19/55 (34.5%) and 21/55 (38.1%) carcinoma cases, respectively. EGFR protein expression was statistically associated with grade (P=0.01), and also with stage (P=0.001) of the examined tumors. Borderline statistical significance was assessed correlating overall cyclin D1 expression to h TERT expression (P=0.06). Simultaneous up regulation of the three proteins was established in 7/55 (12.7%) cases, correlated to the stage of the tumors (P=0.05). EGFR gene amplification was observed in 7/65 (10.7%) carcinomas and dysplasias, whereas chromosome 7 aneuploidy was detected in 4/65 (6.1%) of those cases.Simultaneous up regulation of EGFR, cyclin D1 and h TERT proteins correlates with advanced stage in LSCC. EGFR gene amplification and not only protein over expression maybe is the eligible criterion for targeted therapeutic strategies in those patients.

Presence of Serum Antibodies to Influenza A Subtypes H5 and N1 in Swans and Ibises in French Wetlands, Irrespective of Highly Pathogenic H5N1 Natural Infection

Highly pathogenic (HP) avian influenza A viruses (AIVs) subtype H5N1 (subclade 2.2) were detected in wild birds during outbreaks in France during winter 2006 and summer 2007 in la Dombes wetlands (eastern France) and in Moselle wetlands (northeastern France), respectively. Blood samples from apparently healthy wild birds were collected in 2006 and 2007 from the end of the outbreak to several weeks after the influenza A outbreak inside and outside the contaminated areas, and in 2008 outside the contaminated areas. The samples were tested for the presence and/or quantitation of serum antibodies to influenza A subtypes H5 and N1 using hemagglutination inhibition tests (HITs), a commercial N1-specific enzyme-linked immunosorbent assay kit, and virus neutralization assay. In the HIT, low pathogenicity (LP) and HP H5 AIVs (belonging to H5N1, H5N2, and H5N3 subtypes) were used as antigens. One hundred mute swans were bled in the la Dombes outbreak area in 2006. During 2007, 46 mallards, 69 common pochards, and 59 mute swans were sampled in the Moselle outbreak area. For comparison, blood samples were also collected in 2007 from 60 mute swans from the Marne department where no HP H5N1 influenza A cases have been reported, and in 2008 from 111 sacred ibises in western France where no HP H5N1 influenza A infections in wild birds have been reported either. Mute swans (irrespective of their origin and time of sampling) and sacred ibises (from an area with no known outbreaks) had the highest prevalence of positive sera in the H5 HIT (49-69% and 64%, respectively). The prevalence of anti-H5 antibodies in mallards and common pochards was lower (28% and 27%, respectively). Positive H5- and N1-antibody responses were also significantly associated in swans (irrespective of their origin and time of sampling) and in sacred ibises. However, in swans from the area without outbreaks, the HIT titer against an H5N1 LPAIV was significantly higher than against an H5N1 2.2.1 HPAIV, whereas no difference could be shown for swans from the outbreak areas sampled in 2006 and 2007. These results suggest that ibises and swans from areas without declared outbreaks had acquired humoral immunity after AIV infections with subtypes H5 and N1 but independently from HP H5N1 infection. However, for swans living in outbreak areas, it cannot be excluded that this immunity might result from either a subclinical or a nonlethal infection by HP H5N1.

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.