Child sexual abuse presenting to District General Hospital, Chilaw

Introduction: Incidence of child sexual abuse is increasing worldwide. There is little data on child sexual abuse in the North Western Province of Sri Lanka. Objectives: To describe the demographic and medico-legal findings of victims of sexual abuse aged less than 16 years and to identify factors associated with positive findings on medical-legal examination. Methods: This cross-sectional analytical study, analysed 132 victims, referred by authorities to General Hospital,Chilaw, from 2012 to 2014. Results: Mean age of the victims was 13.1years. Main types abuse were penetrative (61.4%), intra-crural (39.4%) and anal sex (10.6%). The perpetrator was known in 94%. Places of offence were offender's habitat (55.3%), victim's home (23.5%) and secluded areas (11.4%). A positive history of penetrative sex (OR =15.3; 95% CI 5.6-42), chronic sexual abuse (OR=4.8; 95% CI 2.2-10.5) and time lapse in reporting to authorities (OR=4.4; 95% CI 2.0-9.4) were significantly associated with presence of conclusive medical examinations findings. Children were less likely to be willing partners in intra-familial abuse compared to extra-familial abuse where the child was sometimes a willing partner, (OR=0.2; 95% CI 0.07-0.5). Adverse psychological outcomes were observed in 16.7% (n=22). Conclusions: Most children were victims of statutory rape and knew the perpetrator. In extra-familial abuse, child was sometimes a willing partner. A positive history of penetrative sex, number of abusive incidents and time lapse for presentation were important factors associated with conclusive medical findings.

An improved method for efficient recovery of high quality DNA from date palm (Phoenix dactylifera L; Arecaceae).

Date palm (Phoenix dactylifera L; Arecaceae) is one of a few fruit trees that can remarkably grow in dessert agroecosystems that are characterized by extreme temperature fluctuations. Due to increasing demands for dates in the global market and commercial cultivation in many countries, the tree is currently under extensive research in many countries, particularly to improve the germplasm using different molecular tools. Most molecular techniques largely depend on good quality DNA in significant quantities, which are highly compromised by the presence of various contaminants in DNA. The traditional cetyltrimethylammoniumbromide (CTAB) based method has failed to produce good quality DNA from date palm due to hard fibrous nature of tissue. On the basis of previous studies, commercial DNA extraction kits are not economical although they are very effective. Therefore, we have developed an improved DNA extraction protocol by modifying the original CTAB method to produce extra pure DNA in large quantities. The novel method has been validated using different quality testing approaches. This cost-effective method can be used successfully for DNA extraction from date palm. Moreover, this improved method may have potential for DNA extraction from other palms that have similar leaf texture to date palm leaves, but this method needs to be tested for other palms before being used. The improved method has following key modifications:•Grinding of plant tissue in liquid nitrogen and subsequent lysis of cells in CTAB buffer that has increased concentration of ß-mercaptoethanol•Repeated steps of chloroform: IAA extraction and ethanol washing•Addition of RNase A before the DNA precipitation step.

Current Aspects of the Endocannabinoid System and Targeted THC and CBD Phytocannabinoids as Potential Therapeutics for Parkinson's and Alzheimer's Diseases: a Review.

Neurodegeneration leading to Parkinson's disease (PD) and Alzheimer's disease (AD) has become a major health burden globally. Current treatments mainly target controlling symptoms and there are no therapeutics available in clinical practice to preventing the neurodegeneration or inducing neuronal repairing. Thus, the demand of novel research for the two disorders is imperative. This literature review aims to provide a collection of published work on PD and AD and current uses of endocannabinoid system (ECS) as a potential drug target for neurodegeneration. PD is frequently treated with L-DOPA and deep brain stimulation. Recent gene modification and remodelling techniques, such as CRISPR through human embryonic stem cells and induced pluripotent stem cells, have shown promising strategy for personalised medicine. AD characterised by extracellular deposits of amyloid ß-senile plaques and neurofibrillary tangles of tau protein commonly uses choline acetyltransferase enhancers as therapeutics. The ECS is currently being studied as PD and AD drug targets where overexpression of ECS receptors exerted neuroprotection against PD and reduced neuroinflammation in AD. The delta-9-tetrahydrocannabinoid (Δ9-THC) and cannabidiol (CBD) cannabinoids of plant Cannabis sativa have shown neuroprotection upon PD and AD animal models yet triggered toxic effects on patients when administered directly. Therefore, understanding the precise molecular cascade following cannabinoid treatment is suggested, focusing especially on gene expression to identify drug targets for preventing and repairing neurodegeneration.

Use of bovine myeloperoxidase as an indicator of mastitis in dairy cattle.

Bovine myeloperoxidase (MPO) is a heme protein consisting of both large and small polypeptide subunits. In mammals the role of MPO in defending against microbes is well documented. To evaluate the potential of using MPO in the diagnosis of udder infections in dairy cattle we developed a specific enzyme immunoassay for bovine MPO in milk. Antibodies against bovine MPO were produced using the purified enzyme. The ELISA utilizes two specific antibodies: one that is anti-MPO monoclonal and one that is anti-MPO polyclonal. For a total of 141 milk samples the correlation coefficient between the somatic cell count and MPO concentrations determined using the ELISA was 0.91. The ELISA showed good precision and accuracy in measuring MPO in milk, with a total variation of ca. 10%. The recoveries of known amounts of MPO from milk were satisfactory. Thus the stability of the enzyme in milk was judged to be good. Microorganisms were isolated in ca. 85% of the milk samples with elevated concentrations of MPO. Microorganisms were not isolated from more than 90% of non mastitic milk samples with low somatic cell counts where MPO was not detectable using ELISA. The results clearly show that the quantitative analysis of the amount of MPO in mastitic milk can be used to detect intramammary infections in dairy cattle.

Bactericidal activity of the bovine myeloperoxidase system against bacteria associated with mastitis.

Myeloperoxidase (MPO) is a lysosomal enzyme found in the primary granules of mammalian neutrophils. Together with MPO, peroxide and halide form a system of defense against bacteria. The present investigation was undertaken to study the bactericidal effects of the bovine-MPO/peroxide/halide system on pathogenic bacteria associated with bovine mastitis. We demonstrated that MPO together with oxidizing agents generated by xanthine oxidase, hypoxanthine and chloride form a potent antibacterial system against the common udder pathogens Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli in a synthetic medium. However, when milk was added to the reaction mixture, the bactericidal properties of this enzyme system were completely inhibited. Loss of bactericidal activity in the milk-containing cultures was unable to be restored by increasing the concentration of MPO. Nor did an increase in concentrations of hypoxanthine and xanthine oxidase, or the replacement of the above-mentioned peroxidase generating system with a high concentration of hydrogen peroxide, significantly elevated the bactericidal activity that was inhibited by milk. The addition of bovine serum albumin to the synthetic medium reduced the bactericidal activity of the MPO/peroxide/chloride system in a dose-dependent manner. Therefore, milk proteins probably form adducts with strong bactericidal agents that are generated by the MPO system and thereby reduce the bactericidal potential of this system.

Formylmethionyl-leucyl-phenylalanine-induced chemiluminescence responses in bovine polymorphonuclear leukocytes.

Isolated bovine polymorphonuclear leukocytes (PMNL) show a biphasic luminol-dependent chemiluminescence response, and when exposed to high concentrations (2 x 10(-4) M) of the chemotactic peptide formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe), the second emission peak is enhanced. This response was increased in magnitude by incubating PMNL at room temperature before stimulation with f-Met-Leu-Phe. Pre-exposure of bovine PMNL to myeloperoxidase (MPO) also enhanced their receptiveness to the chemotactic peptide, indicating that the MPO-hydrogen peroxide system can modulate the bovine PMNL response to chemotactic factors. The results demonstrate that bovine PMNL are stimulated by the chemotactic peptides to participate in inflammatory processes.

Casein effects on the myeloperoxidase-mediated oxygen-dependent bactericidal activity of bovine neutrophils.

Effects of casein on the respiratory burst and bactericidal activity of bovine neutrophils were studied in vitro using chemiluminescence measurements of neutrophils and the myeloperoxidase (MPO)/peroxide/chloride bactericidal system in neutrophil-free cultures. At physiological concentrations, casein inhibited the respiratory burst responses of unstimulated bovine neutrophils and those stimulated with opsonized zymosan or phorbol-12-myristate-13-acetate (PMA). The inhibition did not result from impaired activation of neutrophils by the phagocytic stimuli, reduced light transmission or cellular toxicity. Neutrophil respiratory burst activity and the bactericidal activity associated with this phenomenon, i.e. the MPO/peroxide/chloride bactericidal system, were inhibited by casein through the modification of the oxygen radical generating pathway or oxygen radical scavenging. It has long been known that milk inhibits the bactericidal activity of neutrophils. However, the previously undocumented mechanism of action of the major milk protein requires further examination in order to determine whether the neutrophil oxygen-dependent bactericidal pathway plays an important role in defending the lactating bovine udder against bacteria causing mastitis.

Isolation and purification of bovine myeloperoxidase from neutrophil granules.

Bovine myeloperoxidase (MPO) was isolated and purified from neutrophil granules using protein extraction at pH 4 and gel filtration combined with fast protein liquid chromatography. The extracted protein was identified as MPO based on its absorption spectrum, amino acid composition, peroxidase activity and polypeptide structure. Bovine neutrophils contained three different forms of MPO (I, II and III). When subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis each of the three purified forms showed two distinct bands corresponding to heavy and light polypeptide chains of approximately 57,000 and 15,000 molecular radius. Amino acid analysis of the three forms showed that there was an overall similarity between them. Slight differences were found between MPO Form III and the other two forms. The three forms of bovine MPO were shown to differ in their specific enzyme activities in a luminol-dependent chemiluminescence assay. MPO Form III showed the highest enzyme activity. The amount recovered during purification of the respective MPO forms varied, with the recovery being highest for MPO I. Our findings suggest that there are intrinsic differences between the three forms of bovine MPO. In terms of their amino acid composition and molecular weight, the bovine MPO closely resembled human and canine MPO.

Preparation and characterization of monoclonal antibodies against bovine myeloperoxidase.

We established eight cloned B-cell hybridomas producing monoclonal antibodies (Mo abs) against bovine myeloperoxidase (MPO). These anti-MPO (AM) Mo abs, designated AM1-AM8, all reacted similarly to three chromatographic forms of MPO, isolated from a single donor, in an enzyme linked immunosorbent assay. According to immunoblot analysis and ELISA the AM Mo abs are specific to bovine MPO and show no cross reactivity with other neutrophil granule proteins such as lactoferrin, lactoperoxidase and serum albumin. In immunoblot analyses IgG1 class AM1, AM2, AM3 and AM4 Mo abs immunostained the heavy subunit of the MPO (57 kDa). Additionally, the AM Mo abs seem to bind either the reactive site or epitopes on bovine MPO that affect the peroxidase activity of this enzyme. AM Mo abs reacted specifically with neutrophils but did not react with lymphocytes or epithelial cells. The present study shows that these AM Mo abs could be used for developing immunoassays to measure bovine MPO from biological fluids and for localizing neutrophils at sites of infections. They could also be useful in studies assessing the involvement of MPO in inflammatory processes in bovine species.

Effects of some mycotoxins on mitogen-induced blastogenesis and SCE frequency in human lymphocytes.

The effects of T-2 toxin, diacetoxyscirpenol, ochratoxin A and zearalenone on DNA synthesis in phytohaemagglutinin-stimulated human peripheral blood lymphocytes were studied by assaying the incorporation of [3H]thymidine. Total inhibition was obtained with 8 ng T-2 toxin/ml, 8 ng diacetoxyscirpenol/ml or 30 micrograms zearalenone/ml, and with 20 micrograms ochratoxin A/ml inhibition was almost complete; 50% inhibition was produced by 1.5 ng T-2 toxin/ml, 2.7 ng diacetoxyscirpenol/ml, 14 micrograms zearalenone/ml or 14 micrograms ochratoxin A/ml. The toxicity of the trichothecenes to the lymphocytes was slightly reduced when rat liver cells were present whereas the toxicity of ochratoxin A and zearalenone was unaltered. Low concentrations of trichothecenes did not produce any inhibition of DNA synthesis when the cultures were washed and placed in fresh media containing only phytohaemagglutinin 4 hr after the addition of the test compounds. Sister chromatid exchange frequency was not elevated by T-2 toxin, diacetoxyscirpenol or ochratoxin A. Zearalenone had a weak enhancing effect on the frequency of sister chromatid exchanges.

Modulation of resistance to mastitis pathogens by pretreatment of mice with T-2 toxin.

T-2 toxin, a secondary metabolite of Fusarium species, is a mycotoxin with immunomodulatory activity. In the present investigation the effects of T-2 toxin on host resistance was studied. The virulence of Escherichia coli and Staphylococcus aureus in the mammary glands of mice treated with T-2 toxin was compared with their virulence in control mice. Virulence was estimated from the ability to induce various types of lesions and bacterial growth in the mammary gland. Pretreatment of mice with a single dose (3 mg/kg body weight) of T-2 toxin by gavage reduced the virulence of both E. coli (P less than 0.05) and S. aureus (P less than 0.01). Microscopic lesions in the infected glands varied in character, from consistently non-reactive necrosis of the entire mammary gland to limited inflammatory reactions. The former were more abundant in control mice than in mice treated with T-2 toxin. Although treatment by gavage with T-2 toxin (0.75 mg/kg body weight/day) for 14 days prior to inoculation had no significant effect on the course of the mastitis infection, virulence was slightly lower in the T-2 toxin treated mice. Both single-dose and successive treatment with T-2 toxin enhanced the respiratory burst activity of macrophages. Pre-inoculation treatment with T-2 toxin also caused a significant increase in the number of peritoneal cells, T-2 toxin did not show bacterial effects on the E. coli or S. aureus strains used for the inoculations. The data indicate that T-2 toxin has modulatory effects on the cell-mediated immune system, and that enhancement of resistance to common mastitis pathogens in mice pretreated with a single dose of T-2 toxin is associated with migration and activation of macrophages.

Effect of T2 toxin on the spontaneous antibody-secreting cells and other non-lymphoid cells in the murine spleen.

The Fusarium secondary metabolite, T2 toxin was investigated for its effects upon spontaneous antibody-producing cells in the murine spleen. Changes in the number of non-lymphoid cells in the spleen were also studied. A statistically significant increase in the number of spontaneous antibody-secreting cells, as detected by the protein A plaque assay, was observed. A dose-dependent increase was found after chronic exposure to T2 toxin for 3 wk. An increased number of erythroblast cells was also found in the spleens of these animals. At all the T2 toxin concentrations studied, the mice showed a dose-dependent reduction in blood levels of haemoglobin. Reduction in the suppression of B cell growth and stimulation of B cells caused by erythropoiesis or activated macrophages could be responsible for the increase in antibody production.

The effects of patulin and patulin-cysteine mixtures on DNA synthesis and the frequency of sister-chromatid exchanges in human lymphocytes.

The effects of patulin and patulin-cysteine mixtures on DNA synthesis in human blood lymphocytes were measured by assaying incorporation of [3H]thymidine into cellular DNA. Patulin inhibited and patulin-cysteine mixtures stimulated the incorporation of [3H]thymidine into DNA. The inhibitory action of patulin diminished with time in culture. Patulin was found to be unstable in the culture medium. The sister-chromatid exchange (SCE) frequency was significantly elevated by intermediate concentrations (0 . 1-0 . 2 micrograms/ml culture) of the toxin. The cells are protected from the effect at low concentrations of the toxin. There may be excessive damage at higher concentrations but only the unaffected cells go into mitosis. Therefore an increased frequency of SCEs is detected only at intermediate concentrations, or, at higher concentrations, with early harvesting. Cysteine seems to potentiate the effect of patulin on SCE frequency.

Multicenter analytical evaluation of a high-sensitivity troponin T assay.

BACKGROUND: High-sensitivity cardiac troponin assays are being introduced clinically for earlier diagnosis of acute myocardial infarction (AMI). We evaluated the analytical performance of a high-sensitivity cardiac troponin T assay (hscTnT, Roche Diagnostics) in a multicenter, international trial. METHODS: Three US and 5 European sites evaluated hscTnT on the Modular® Analytics E170, cobas® 6000, Elecsys 2010, and cobas® e 411. Precision, accuracy, reportable range, an inter-laboratory comparison trial, and the 99th percentile of a reference population were assessed. RESULTS: Total imprecision (CVs) were 4.6-36.8% between 3.4 and 10.3 ng/L hscTnT. Assay linearity was up to 10,000 ng/L and the limit of blank and detection were 3 and 5 ng/L, respectively. The 99th percentile reference limit was 14.2 ng/L (n=533). No significant differences between specimen types, assay incubation time, or reagent lots existed. A substantial positive bias (76%) exists between the 4th generation and hscTnT assays at the low end of the measuring range (<50 ng/L). hscTnT serum pool concentrations were within 2SD limits of the mean of means in the comparison trial, indicating comparable results across multiple platforms and laboratories. CONCLUSION: The Roche hscTnT assay conforms to guideline precision requirements and will likely identify additional patients with myocardial injury suspicious for AMI.

Defective polymorphonuclear neutrophil function in dairy cows showing enhanced susceptibility to intramammary infections.

Polymorphonuclear-neutrophil (PMN) oxidative-burst activity, chemotactic and chemokinetic migratory responses, and surface-adhesion protein expression in a mastitis-prone group of dairy cows were compared with corresponding variables in healthy cows. The cows had a well-documented history of udder infection caused by major mastitis pathogens. Analysis of PMN functions revealed a deficiency in the luminol-enhanced chemiluminescence responses that seemed to be associated with the mobilization of myeloperoxidase (MPO) in the PMN of the patient group, as compared with the healthy controls. The migratory capacity of the PMN in response to a variety of chemotactic substances was enhanced in the patients. However, there were no significant differences between the two groups in the expression of surface-adhesion proteins (CD11a/CD18). It is proposed that the migratory activity of PMN cells was enhanced in order to compensate for their depressed respiratory-burst activity. Studies are under way to assess whether the defective mobilization of MPO in PMN of mastitis-prone cows is an acquired transient defect or a permanent hereditary defect.

The ultrastructure of cells and cell fragments in mammary secretions of Camelus bactrianus.

The fine structure of leucocytes from the udders of 7 lactating and 3 non-lactating bactrian camels and from peripheral blood were studied. The most important finding was the presence of large numbers of cell fragments in milk. The cell fragments were bounded by a plasma membrane, had no nuclei and contained mitochondria and abundant rough endoplasmic reticulum (RER). Macrophages were the dominant cells recovered from milk and udder washings during the dry period. Neutrophils and lymphocytes were also present. The dominating leucocytes in blood were neutrophils, followed by lymphocytes, eosinophils, monocytes and basophils.

Ginseng-enhanced oxidative and phagocytic activities of polymorphonuclear leucocytes from bovine peripheral blood and stripping milk.

This study investigated the effect of a dried ginseng extract on polymorphonuclear leucocytes (PMNL) in bovine blood and milk. In a test for chemiluminescence (CL), PMNL were pre-incubated in ginseng solution at 37 degrees C in 5% CO2 for 60 min, and then stimulated with bovine serum opsonized zymosan. The CL was about 30% higher for the cells pre-treated with ginseng solutions 100-1,000 micrograms/ml as compared with the non-ginseng-treated cells. In a test for phagocytosis, PMNL and fluorescent microspheres were incubated with ginseng in RPMI-1640 supplemented with 10% bovine serum at 37 degrees C for 60 min. The proportion of actively phagocytic cells in the ginseng-treated group was greater than that in the non-ginseng treated group.

A sensitive double-antibody enzyme-linked immunosorbant assay for bovine myeloperoxidase and its application to serum and neutrophil extracts.

The heme enzyme myeloperoxidase (MPO), with a spectral A430/A280 ratio > 0.78, was purified from isolated bovine neutrophils. Using highly specific anti-MPO monoclonal and anti-MPO polyclonal antibodies raised against MPO, a specific and sensitive double-antibody enzyme-linked immunosorbant assay (ELISA) was developed to measure bovine MPO in serum and neutrophil extracts. The ELISA shows good precision and accuracy, with intra- and interassay coefficients of variation of < 10% for MPO concentrations ranging from 0.5 to 50 ng/ml. The accuracy of the ELISA for measuring MPO in bovine serum was further confirmed by the similarity between the standard curve and curves obtained with successive dilutions of MPO-rich serum samples. The mean analytical recovery of MPO from serum was approximately 90%. Long delays between blood sampling and serum preparation were found to affect the level of MPO in the serum. Mean MPO values in the serum of healthy adult cows were 6.5 ng/ml, with a range of 3.5-15.3 ng/ml. In dairy cows with acute mastitis, mean serum MPO values were approximately 30 ng/ml, with a range of 6.0-59.6 ng/ml, and the elevation was markedly higher than the normal values (P = 0.0001). In isolated neutrophils from healthy cattle, MPO concentrations were found to be 7 x 10(-4) ng, with a range of 6.5-8.3 x 10(-4) ng/neutrophil. The ELISA was used to study the distribution of MPO in the bovine neutrophil granules; it was found to be localized to one distinct compartment.