First insight into the population structure of Mycobacterium tuberculosis in Saudi Arabia

This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of M. tuberculosis, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia.     

A Novel Way of Standardization of ICG Lymphangiography Reporting

Background  Indocyanine green (ICG) lymphangiography is being increasingly employed to assess the severity of lymphedema, locate the areas of patent linear lymphatics and dermal backflow and plan treatment. This study suggests a novel method of reporting ICG findings in extremities to enable easy understanding among surgeons and physiotherapists and avoid repeat testing when a patient visits a disparate lymphedema center or clinician. Methods  A reporting protocol was developed in the lymphedema clinic of the plastic surgery department, and patients were asked to bring along the report in every subsequent review. The ICG findings were recorded on the fluorescence imaging system as well. The report was prepared by one and analyzed by two different clinicians without repeating the test on 10 consecutive patients. Results  The interrater reliability of findings in the report was found to be 98.7% among the three clinicians. Conclusion  The reporting system was found to be illustratable and reproducible     

Early acute antibody-mediated rejection of a negative flow crossmatch 3rd kidney transplant with exclusive disparity at HLA-DP

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.     

Improving the Quality of EDTA-treated Blood Specimens from Mice

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Industrial and academic research often require hematology analyses of mouse blood. Consequently, many terminal and nonterminal techniques have become available for blood sampling in mice.^{12,21,27,40,42,53} Preanalytical variation in clinical pathology is known to be a major issue.^5,45,49 Although the effects of the blood sampling method on animal welfare have been the subject of many preanalytical hematology and biochemical analyses,^{1,6,8,9,15,16,18,24-26,36,47,50-52,54} no agreement has been reached regarding the optimal method for nonterminal blood collection in mice and, to our knowledge, only a few investigations^1,8,15,16,18 have addressed the quality of the resulting blood specimens.Our own experience of hematology measurements from nonterminal mouse EDTA-blood specimens is that some specimens show both visible clots and platelet aggregation, the latter being detected only from microscopic examination of blood smears.³³ Whereas specimens with visible clots can be eliminated, microscopic platelet aggregates can also interfere with hematology analyses or cause analytical errors, as has been reported in other species including cats.^13,22,31,39 These abnormalities require repeat sampling when possible; otherwise, the number of validated results is decreased. EDTA-treated mouse blood is especially prone to platelet aggregation and clotting.^14,28,43 This characteristic leads to errors in platelet counts (pseudothrombocytopenia) and possible misidentification of platelet aggregates as eosinophils, resulting in false leukocytosis and eosinophilia.¹⁴ In vitro platelet aggregation in mice is due to high platelet counts^34,43 and is influenced by numerous preanalytical factors including the sampling method, collection site, specimen processing, anticoagulant used, the blood:anticoagulant ratio, the mouse strain and genetic alterations.^19,28,30,43 The literature on the influence of preanalytical factors on the quality of CBC analyses in mice is scant,⁴³ and no agreement has yet been reached regarding the optimal method for nonterminal blood collection in mice. In humans and various animal species, platelet aggregation can be reduced by adding platelet aggregation inhibitors that act at different steps of aggregation. To our knowledge, the addition of such inhibitors to mouse whole blood has not been tested as a means to improve the quality of mice EDTA-treated blood specimens.The aim of this study was therefore to identify the best preanalytical conditions for nonterminal blood collection in mice, based on animal welfare, scores of platelet aggregation, and platelet, RBC, and WBC counts. The hypotheses we tested were that 1) adding an antithrombotic drug (or multiple such drugs) to the EDTA-treated blood specimen would prevent or at least significantly lower platelet aggregation, 2) the site and the method of collection influence in vitro platelet aggregation, and 3) high-quality blood sampling is a key to reducing platelet aggregation in blood specimens.     

Functional characterization of the pentapeptide QYNAD on rNav1.2 channels and its NMR structure

The endogenous pentapeptide QYNAD (Gln-Tyr-Asn-Ala-Asp) is present in human cerebrospinal fluid (CSF), and its concentration is increased in demyelinating diseases. QYNAD was synthesized and its action on the rNa_v1.2 voltage-gated sodium channel -subunit was studied using whole-cell recordings in a heterologous expression system. The effects were seen only upon equilibration of the peptide in the external bath solution for at least 10 min before the commencement of whole-cell experiments. The steady-state activation curve showed a rightward shift of 10 mV, while the steady-state inactivation curve showed a leftward shift of 5 mV. Frequency-dependent inhibition of the sodium current amplitude was observed at 2–10 Hz, in the presence of external QYNAD, but was not seen when applied internally. Fits of the whole-cell sodium current traces by Hodgkin-Huxley equations revealed subtle changes in the voltage-dependent rate constants governing the transition of the activation and the inactivation gates. Two dimensional NMR spectroscopy revealed the absence of medium and long-range Nuclear Overhauser effects (NOEs), which indicates that the peptide does not adopt any canonical secondary structure in solution. In summary, our studies show that although the pentapeptide QYNAD does not have a defined structure in solution, it has defined actions on the rNa_v1.2 voltage-gated sodium channel isoform.     

Ultrasonographic assessment of thyroid volume in Delhi children

BACKGROUND: Ultrasonographic measurement of thyroid volume is increasingly being used to assess prevalence of iodine deficiency in a community. OBJECTIVES: To assess thyroid volume by ultrasonography in normal Delhi children, and compare them with the recently adopted WHO reference standards. METHODS: One hundred and three children aged of 6-10 years were enrolled from the paediatric outpatient clinic in a prospective study. Thyroid volume was estimated by ultrasound, and urinary iodine levels were measured for all subjects by spot urine samples. RESULTS: Goitre prevalence was 24.3% on clinical assessment. Urinary iodine levels fell in the range of mild to moderate deficiency in 61% of the children. None of the children met the WHO recommended ultrasonographic criteria for goitre (thyroid volume >97th percentile of the reference standards), when WHO age-specific norms were used for comparison. On applying body surface area (BSA)-specific norms, only two children were classified as having goitre. CONCLUSIONS: The WHO standards for thyroid volume by ultrasonography tended to underestimate goitre prevalence.     

Removal of iodine by solid phase adsorption to charcoal following iodine oxidation of acetamidomethyl-protected peptide precursors to their disulfide bonded products: oxytocin and a Pre-S1 peptide of hepatitis B virus illustrate the method

Abstract: Chemical quenching, gel filtration or liquid phase extraction procedures are currently in vogue for taking iodine off from the reaction mixtures in which it is used to cause the formation of disulfide bonds in acetamidomethyl or trityl protected peptides. It has been found that charcoal effectively, selectively and rapidly removes iodine by solid phase extraction from reaction mixtures in which it is used to convert the acetamidomethyl protected precursors of oxytocin or a peptide from the Pre-S1 region of hepatitis B virus into their intramolecularly disulfide-bonded products. The advantages of this new method, namely simplicity, rapidity, quantitative yields, freedom from side reactions, linear scalability, cost effectiveness and adsorption of iodine on to solid charcoal are discussed.     

Bioeffective monoclonal antibody against the decapeptide gonadotropin-releasing hormone: reacting determinant and action on ovulation and estrus suppression.

A monoclonal antibody generated against the decapeptide gonadotropin-releasing hormone (GnRH) was effective in intercepting the bioactivity of the hormone; it blocked ovulation in rats. The antibody reacted optimally with the native hormone. Substitution of amide at the COOH terminus by a carboxyl group decreased immunoreactivity by a factor of 200. The antibody recognized the amino acid sequences 4-6, 7-10, and 4-10 to a variable degree, which suggests that the epitope has a conformation involving the entire molecule, with the NH2- and COOH-terminal regions probably in proximity. The antibody was also competent to suppress the progression of estrus in dogs, an indication that GnRH may play an inductive role in the reproductive function of dogs.     

Immunobiological properties of a carboxy-terminal 53-amino acid peptide of the beta subunit of human chorionic gonadotropin

Lengthening of the carboxy terminus unique region of beta-hCG from 30 to 45 amino acids was found in previous studies to improve immunogenicity and hormone neutralization capacity. The present study was carried out to determine whether further elongation of the peptide to 53 amino acids enhances to hormone neutralization capacity without loss of specificity characteristics. The peptide 93--145 of beta-hCG with substitution of cysteines at 93, 100 and 110 by alpha-aminobutyric acid was synthesized by solid phase and conjugated to tetanus toxoid by an active ester method. Rabbit antibodies against this conjugate reacted with CTP-53 and CTP-45 with a parallel slope in anti-CTP-53-[125I]Tyr-CTP--53 radioimmunoassay system. Other CTPs, e.g. CTP-26, CTP-31 and CTP-35 competed with lower efficiency; 50% inhibition of binding was obtained with 10-100 pmol/tube with these peptides instead of 0.5 pmol/tube for the homologous CTP-53. Anti-CTP-53 reacted with both beta-hCG and hCG but around twenty times greater amount of hCG was required to give 50% inhibition of binding as compared to CTP-53. The antigen binding capacity of anti-CTP-53 was around 4000 ng/ml for CTP-53 and 25 ng/ml for hCG. The anti-CTP-53 sera retained non-cross-reactivity with hLH as determined by direct binding with [125I]hLH and by competitive inhibition of CTP-53 binding with anti-CTP-53. Anti-CTP-53 neutralized the bioactivity of hCG in the Leydig cell bioassay and in the mouse uterine weight gain assay. Anti-CTP-53 antibodies were about three times more effective than anti-CTP-45 in their capacity to neutralize the bioactivity of hCG, though still substantially poorer than anti-beta-hCG sera in this respect.