Acute flaccid paralysis (AFP) surveillance intensification for polio certification in Kaduna state, Nigeria: lessons learnt, 2015-2016.

BACKGROUND: Nigeria has made remarkable progress in its current efforts to interrupt wild poliovirus transmission despite the re-emergence of wild poliovirus in 2016. The gains made in Nigeria have been achieved through concerted efforts by governments at all levels, traditional leaders, health workers, caregivers, and development partners. The efforts have involved an elaborate plan, coordination, and effective implementation of routine immunization services, supplemental immunization activities, and acute flaccid paralysis (AFP) surveillance. METHODS: We conducted the following activities to strengthen AFP surveillance in Kaduna state: a monetary reward for all AFP cases reported by health workers or community informants and verified as "true" AFP by a World Health Organization (WHO) cluster coordinator; training and sensitization of surveillance officers, clinicians, and community informants; recruitment of more personnel and expansion of the surveillance network; and the involvement of special populations (nomadic, hard-to-reach, and border communities) and caregivers in stool sample collection. The paired t test was used to evaluate the impact of the different initiatives implemented in Kaduna state to intensify AFP surveillance in 2016. RESULTS: There was increased annualized non-polio AFP rate (ANPAFPR) in 21 out of 23 Local Government Areas (LGAs) of Kaduna state 6 months after implementation of different initiatives to intensify AFP surveillance. The AFP reported by the special population increased in 15 out of 23 LGAs. Statistical analyses of mean scores of ANPAFPR before and after the interventions using the paired t test revealed a significant difference in mean scores: mean = 19.7 (standard deviation (SD) = 16.1) per 100,000 < 15 years old in July-December 2015, compared with 38.0 (SD = 21.6) per 100,000 < 15 years old in January-June 2016 (p < 0.05). Likewise, analysis of silent wards using the paired t test showed a significant difference in mean scores: mean = 4.0 (SD = 2.1) in July-December 2015 compared with 2.4 (SD = 1.8) in January-June 2016 (p < 0.05). CONCLUSION: The different initiatives implemented in 23 LGAs of Kaduna state to intensify AFP surveillance may be responsible for the significant improvement in the AFP surveillance performance indicators in 2016.

[Clinical variability of Best's disease]. / Klinická rozmanitost Bestovej choroby.

Retrospective view of the various phenotypes 20 persons affected by classic solitary form of vitelliform macular dystrophy, in 3 pedigrees with autosomal dominant transmission and in 4 single cases. Long-term monitoring allows to observe the variability of expression, from classic course to peculiarity of the clinical expression in the disc development and their corresponding functions of the central retina.

Size exclusion chromatography as a tool for evaluation of fragmentation pattern of gastric mucins under non-degrading conditions.

Gastric mucins are high molecular weight extracellular glycoproteins that play a major role in the protection of the gastrointestinal tract and besides that they are also involved in many disease processes. In the present study, size exclusion chromatography under non-degrading conditions was used to study the fragmentation pattern of native gastric mucins. The samples of gastric mucins of different origin obtained by an extraction of gastric mucosa with Tris-HCl buffer, pH 7.3 were separated using size exclusion chromatography on Sephadex G-100. While samples of rat gastric mucins are characterized by the presence of only high-molecular weight fraction of glycoproteins, fragmented mucin components in non-denaturated samples were observed in canine and human gastric mucins. Differences in the fragmentation pattern were observed in patients with ulcer diseases and gastric cancer. Degradation products of mucins were also detected using polyacrylamide gel electrophoresis in the presence of SDS.

Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract.

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3-8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.

Origin, localization and binding abilities of boar DQH sperm surface protein tested by specific monoclonal antibodies.

Seminal plasma proteins bind the sperm surface at ejaculation and may modulate several aspects of sperm activity during reproduction. DQH sperm surface protein, present in boar seminal plasma, shows affinity to phoshorylcholine, acidic polysaccharides, oviductal epithelium and zona pellucida glycoproteins. Monoclonal antibodies (MAbs) against DQH protein were prepared and used for determination of the DQH protein origin in boar reproductive organs, its localization on boar spermatozoa, and for investigation of its binding abilities in the porcine oviduct and to the zona pellucida of the oocyte. The mRNA transcript of DQH protein was found in seminal vesicles and not in the testis, epididymis and prostate. Its translated products were immunodetected by MAbs in seminal vesicle extract and fluid, in seminal vesicle tissue sections and on the membrane-associated acrosomal part of ejaculated spermatozoa. These results confirm the ability of DQH protein to bind the sperm surface at ejaculation and to participate in formation of the sperm reservoir in the porcine oviduct. Moreover, monoclonal antibodies reduced binding of sperm to oocytes and proved the role of DQH protein in the sperm-zona pellucida primary binding.

Separation, characterization and identification of boar seminal plasma proteins.

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.

Affinity chromatography of bull seminal proteins on mannan-Sepharose.

The interaction of bull seminal plasma proteins and sperm with mannan was investigated using an enzyme-linked binding assay (ELBA). A high mannan-binding activity was found in the protein fraction interacting with heparin. Mannan binding to seminal plasma proteins was inhibited by D-mannose and D-fructose, but not by D-mannose-6-phosphate, D-glucose-6-phosphate, ovalbumin and ovomucoid. Mannan inhibited the binding of bovine zona pellucida glycoproteins both to bull sperm and seminal plasma proteins. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. The protein components of this fraction were identified on the basis of relative molecular mass determination and N-terminal amino acid sequencing: RNAase dimer, PDC-109 and a protein homologous to BSP-30K (relative molecular mass 14,500). The isolated proteins were characterized by a high zona pellucida binding activity.

Immobilization of L-glyceryl phosphorylcholine: isolation of phosphorylcholine-binding proteins from seminal plasma.

The preparation of an affinity sorbent containing immobilized L-glyceryl phosphorylcholine for affinity chromatography of phosphorylcholine-binding proteins from seminal plasma is described. The ligand was coupled either after its maleinylation to poly(acrylamide-allyl amine) copolymer or directly to divinyl sulfone-activated Sepharose. The prepared phosphorylcholine derivative coupled to Sepharose was used for affinity chromatography of phosphorylcholine-binding proteins from bull and boar seminal plasma. Adsorbed proteins were specifically eluted with phosphorylcholine solution. Isolated phosphorylcholine-binding proteins were characterized by SDS electrophoresis and HPLC with reversed phase. Composition of the boar phosphorylcholine-binding fraction obtained by affinity chromatography on immobilized L-glyceryl phosphorylcholine was compared with that eluted from immobilized heparin by the phosphorylcholine solution. No phosphorylcholine-binding proteins were found in human seminal plasma.

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases.

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.

Heparin-binding proteins of human seminal plasma homologous with boar spermadhesins.

Protein homologues to boar seminal plasma spermadhesins with the N-terminal sequence AQN (AQN spermadhesins) and with the N-terminal sequence AWN (AWN spermadhesins) were detected in human seminal plasma and characterized. They were isolated as heparin-binding (HB) proteins from human seminal plasma by affinity chromatography on heparin-Sepharose and then separated into 12 fractions (HB1-HB12) by RP HPLC or into four major fractions (HB-I-HB-IV) by gel filtration. Rabbit antibody against boar seminal plasma AQN 1 spermadhesin cross-reacted with 10-14 kDa proteins of fraction HB7, and antibody against AWN 1 spermadhesin cross-reacted with 11-14 kDa proteins of fractions HB9 and HB11. Both antibodies interacted with 10-14 kDa proteins in fractions HB-I and HB-II. The N-terminal amino acid sequence (1)AQNKG(5)... was determined in the 14 kDa protein of fraction HB-I cross-reacting with AQN 1 antibodies. A component detected among 10-14 kDa proteins of HB7 cross-reacting with rabbit antiserum against AQN 1 had the N-terminal sequence (1)GELKFVTLVFAVGDYE(16), which is similar to the sequence of a fragment of prostatic acid phosphatase. Lactoferrin and its fragments were immunodetected with rabbit antibody against human milk lactoferrin in fractions HB7-HB11. This was proved by N-terminal sequencing of a lactoferrin fragment immunodetected in fraction HB7. N-terminal amino acid sequence analysis of the dominant component of fraction HB2 revealed the presence of a fragment of semenogelin I.

D-fructose-binding proteins in bull seminal plasma: isolation and characterization.

The heparin-binding activity of bull seminal plasma proteins was inhibited by D-fructose, D-glucose, inulin and glycogen; D-galactose, dextran and mannan had no effect. While the ejaculated sperm-heparin interaction was not influenced by the presence of saccharides, the heparin-binding activity of epididymal sperm was inhibited by D-fructose. The results of the binding studies were confirmed by affinity chromatography on immobilized heparin followed by elution with monosaccharides. Proteins adsorbed to a heparin-polyacrylamide column and eluted with D-fructose were analyzed by RP HPLC, SDS electrophoresis and by determination of the N-terminal amino-acid sequence. RNAase dimer, PDC-109 and metalloproteinase inhibitor (TIMP-2) were identified.

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine.

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.

Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis.

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.

Aggregated and monomeric forms of proteins in boar seminal plasma: characterization and binding properties.

Boar seminal plasma was separated into five protein fractions (I-V) (>100, 55, 45, 30, 5-15 kDa) by gel filtration chromatography on Sephadex G-75 SF at pH 7.4. RP HPLC of protein fractions I-V and N-terminal sequencing of their individual components revealed that high-molecular-weight aggregates consisted mainly of DQH sperm surface protein and AQN, AWN, PSP II spermadhesins, while fraction IV consisted of heterodimers of PSP spermadhesins only. Spermadhesins as monomers were present in seminal plasma in a very low amount. Biotinylated fractions I-IV containing AWN, AQN, DQH, and PSP proteins were bound to boar epididymal and ejaculated spermatozoa with the same efficiency. Aggregates containing AWN, AQN, DQH, PSP II proteins (fractions I-III) and their HPLC-separated monomeric forms interacted with phosphorylcholine. Aggregates containing the DQH protein and AWN spermadhesins as well as their separated monomeric proteins interacted strongly with acidic polysaccharides. PSP II interacted with some acidic polysaccharides, while the fraction IV corresponding to heterodimer PSP IPSP II did not show any binding to acidic polysaccharides and zona pellucida. Fractions I-III showed affinity to cholesterol. The strongest interaction was observed between biotinylated glycoproteins of porcine zona pellucida and AWN 1-containing aggregates and separated proteins. AQN 1 spermadhesin effectively blocked the sperm binding to oocytes. These results suggest that under physiological conditions, the aggregated forms of seminal plasma proteins (DQH, AQN, AWN, PSP II) rather than the individual proteins might take part in coating the sperm surface, in sperm capacitation and in primary binding of spermatozoa to zona pellucida of the ovum.

Sperm surface proteins in mammalian fertilization.

Boar seminal plasma was separated into five protein fractions (I-V) (> 100, 55, 45, 30, 5-15 kDa) by gel chromatography on Sephadex G-75 SF at pH 7.2. RP-HPLC of protein fractions I-V and N-terminal sequencing of their individual components revealed that the high-molecular-weight aggregates consisted mainly of DQH sperm surface protein and AQN, AWN, PSP II spermadhesins, whereas fraction IV consisted of heterodimers of PSP spermadhesins only. Spermadhesins as monomers were present in seminal plasma in a very low amount. Aggregates containing the DQH protein and AWN spermadhesins as well as HPLC-separated monomeric proteins interacted strongly with acidic polysaccharides. The strongest interaction was observed between biotinylated glycoproteins of porcine zona pellucida and AWN 1-containing aggregates and separated proteins. PSP II interacted with some acidic polysaccharides, whereas the fraction IV corresponding to heterodimer PSP I/PSP II did not show any binding to acidic polysaccharides and zona pellucida. Aggregates containing AWN, AQN, DQH, PSP II proteins, and their separated monomeric forms (fractions I-III) interacted with phosphorylcholine. Fractions I-III showed affinity to cholesterol. Biotinylated aggregates containing AWN, AQN, DQH, and PSP proteins (fractions I-IV) bound stronger to boar epididymal spermatozoa than to ejaculated spermatozoa. These results suggest that under physiological conditions, the aggregates of seminal plasma proteins (DQH, AQN, AWN, PSP II) rather than the individual proteins might take part in coating the sperm surface, in sperm capacitation, and in primary binding of spermatozoa to zona pellucida of the ovum.

Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin-resistant boar seminal plasma O-glycoprotein related to pB1 protein.

The complete covalent structure of a novel boar DQH sperm surface protein resistant to many classical procedures of enzymatic fragmentation was determined. The relative molecular mass of the major form of this protein determined by ESI-MS and MALDI-MS was 13,065.2+/-1.0 and 13,065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N-acetylhexosamine), 146 Da (i.e., plus deoxyhexose), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protein was determined using automated Edman degradation, MALDI-MS, and post-source decay (PSD) MALDI-MS, and shown to consist of N-terminal O-glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O-glycosidically linked to threonine 10, as confirmed by PSD MALDI-MS of the isolated N-terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALDI-MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent structure of proteins, especially those that are refractory to standard approaches of protein chemistry.

Aggregated forms of heparin-binding and non-heparin-binding proteins of boar seminal plasma and their binding properties.

Boar seminal plasma proteins were separated by affinity chromatography on immobilized heparin into two portions: heparin-binding (H+) and non-heparin-binding (H-) proteins. Gel chromatography of the H+ portion yielded four main protein fractions of >150, 45, 30 and 20 kDa, while that of the H- portion resulted in the separation into three main protein fractions of >150, 30 and 20 kDa. HPLC analysis and N-terminal sequencing used to characterize the composition of the protein fractions obtained by gel chromatography revealed that all consisted of low (12-16 kDa) molecular weight components: the H+ fraction consisted of DQH sperm protein, AQN and AWN spermadhesins whereas the H- fraction consisted of PSPI and PSPII spermadhesins. The high molecular weight values of fractions obtained by gel chromatography thus suggest that the proteins are present in boar seminal plasma in the form of aggregates. Interactions of individual boar seminal plasma proteins and their aggregates present in the H+ and H- fractions with acid polysaccharides were estimated.

Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma.

Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.

Saccharide-binding properties of boar AQN spermadhesins and DQH sperm surface protein.

Heparin-binding proteins BHB 2-BHB 5 were purified from boar seminal plasma by affinity chromatography on a heparin-polyacrylamide column and reversed phase HPLC. Three of the proteins, BHB 3-BHB 5, were found to be identical to spermadhesins AQN 1-AQN 3 isolated from boar spermatozoa. The lectin-like properties of the isolated proteins BHB 2-BHB 5 were studied using double-diffusion in agarose gel, enzyme-linked binding assay, and inhibition assays of erythroagglutinating activity. It was found that proteins BHB 3-BHB 5 (spermadhesins AQN 1-AQN 3) interacted with glycoproteins containing O-glycosidically bound oligosaccharide chains, but not with those containing only N-linked carbohydrate chains. The strongest interaction was observed between BHB 3 (AQN 1) and desialyzed bovine submaxillary gland mucin, the glycoprotein containing only O-glycosidically linked saccharides. No interaction of BHB 3-BHB 5 proteins with simple saccharides, their derivatives or acidic polysaccharides was observed. Both the hemagglutinating activity and saccharide-binding properties of BHB 2 protein were quite different. Agglutinating activity of human erythrocytes by BHB 2 protein was significantly higher than that by BHB 3-BHB 5 proteins (AQN spermadhesins). In contrast to AQN spermadhesins, BHB 2 protein (DQH sperm surface protein) interacted strongly with acidic polysaccharides and sialyzed glycoproteins, but no binding of desialyzed glycoproteins as well as N-acetyl-alpha-D-galactosaminyl-O-serine,simple monosaccharides and amino sugars was observed.

Interaction of bull, stallion and boar seminal plasma proteins and sperms with acidic polysaccharides.

The interaction of seminal plasma proteins, sperms and detergent-released sperm proteins of three species with different types of acidic polysaccharides was studied. Heparin-binding activity of boar, bull and stallion seminal plasma proteins, sperms and sperm proteins was compared with their ability to interact with polysaccharides differing in the presence of the sulfate groups or in their saccharide moiety (chondroitin sulfate, dextran sulfate, fucoidan, hyaluronic acid). Bull seminal plasma proteins were characterized by higher affinity to heparin, fucoidan and dextran sulfate, while significant differences between different types of polysaccharides were detected in the case of boar proteins. Sperm protein interactions with acidic polysaccharides in bull and stallion were analogous to the binding of seminal plasma proteins. Bull and stallion seminal plasma proteins inhibited the interaction of corresponding sperm proteins with acidic polysaccharides.