Water is an important transmission route for cryptosporidiosis, with at least 165 waterborne outbreaks of cryptosporidiosis documented. Cryptosporidium can be controlled through water treatment by physical removal and UV disinfection, and a method that can determine whether individual oocysts in a routine sample exposed to UV irradiation have been disinfected is of benefit as it offers increased confidence to water operators. The major effects of UV radiation on cell membranes are alterations of proteins, particularly protein cross-linking. UV-B radiation progressively inhibits protein synthesis. Specific free radical scavengers protect cells against killing and inhibition of protein synthesis by UV-B. UV light also crosslinks the complementary strands of DNA and causes the formation of single strand breaks and pyrimidine dimers. The major lesions induced are cyclobutyl pyrimidine dimers (CPDs; also known as thymine dimers, TD). UV-induced DNA lesions in living cells and in some microorganisms can be repaired by the enzyme-dependent nucleotide excision repair (NER), also named dark repair, and the light-dependent reaction known as photoreactivation (PHR). Dark repair and PHR enable UV-inactivated microorganisms to recover and may reduce the efficiency of UV inactivation. Cryptosporidium parvum oocysts are inactivated at 3-40 mJ/cm2 using medium- and low-pressure UV light. Cryptosporidium parvum can undertake photoreactivation and dark repair at the genomic level and NER repair genes have been identified in C. parvum and C. hominis. However, UV inactivation of Cryptosporidium oocysts is irreversible, despite the presence of the UV repair genes. We investigated the following hypotheses for developing a method to demonstrate UV inactivation of Cryptosporidium oocysts: (i) UV disinfection induces the production of reactive oxygen species (ROS); (ii) UV disinfection induces apoptosis; and (iii) UV disinfection causes damage to DNA, which is detectable using fluorogenic DNA reporters. We developed assays for determining UV inactivation of Cryptosporidium oocysts which would remain compatible with, and integrated as much as possible with, existing UK and USA detection methodologies. Our development of suitable methods which can detect UV damage in individual organisms reliably and reproducibly was driven by a search for fluorogenic reporters which could enter and stain UV-killed and damaged Cryptosporidium oocysts. Antioxidants reduce ROS production and the antioxidant glutathione (GSH) plays a significant role in inhibiting the generation of mutagens by ionizing radiation. The presence of GSH, as a putative reporter of UV damage caused by the production of ROS, was investigated in intact, untreated C. parvum oocysts and sporozoites within intact UV-irradiated oocysts (40 mJ/cm2) using the fluorogenic vital dye monochlorobimane (MCB) to detect both GSH levels and activity in oocysts. MCB fluorescence localized GSH in purified, intact, recently excreted and aged C. parvum oocysts, at several nuclear and cytoplasmic sporozoite foci (n=2-6). We did not demonstrate the function of GSH as an endogenous free radical scavenger in UV-irradiated oocysts, and other free radical scavengers are more active than GSH in UV-treated C. parvum oocysts. MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts. The DNA intercalating dye YO-PRO1 (YP) has been used to determine apoptosis and was used to investigate the role of UV irradiation in inducing programmed cell death/apoptosis. YP detected DNA damage in UV-treated (40 mJ/cm2) C. parvum oocysts. YP was incorporated into sporozoite DNA of intact, irradiated oocysts (possibly apoptotic) which exhibited no apparent oocyst wall damage. However, control oocysts did not exclude YP entirely. YP is unlikely to provide a reliable estimate of the possible apoptotic changes that can occur in irradiated oocysts. An antibody raised against TDs (α-TD) was used to identify changes induced by UV light in C. parvum sporozoites and oocysts, and its nuclear location was validated by co-localization with DAPI. A freeze-thawing (five cycles) procedure improved α-TD antibody labelling within irradiated C. parvum oocysts. No α-TD localization was seen in non-irradiated oocysts. Both C. parvum and C. hominis oocysts exposed to different doses of UV light (range, 10-40 mJ/cm2) demonstrated TD lesions following irradiation. We conclude that an immunofluorescence assay using α-TD antibodies which, for C. parvum, has been validated against a neonatal mouse infectivity assay, is suitable for detecting thymine dimers in air-dried oocysts and air-dried sporozoites of C. parvum and C. hominis oocysts, and that the α-dsDNA antibody is a good candidate for a positive control for the assay.
Summary. The uptake of [5-3H]uridine into RNA and DNA of the cells of the uterine luminal epithelium, stroma and myometrium of the rat has been studied in early pregnancy using a technique for separation of the cell fractions before quantitative analysis. Comparisons of the metabolism between the pregnant and pseudopregnant horn of the unilaterally ovariectomized rat has shown that RNA and DNA synthesis are markedly increased by 04.00 hours on the morning of Day 5 in the pregnant horn. This increased metabolism occurs in all cell fractions and before the zona pellucida is shed. The results are discussed in relation to the onset of the decidual response.
Four tricyclic antidepressants, amitriptyline, imipramine, desipramine and iprindole have been shown to partially protect mouse brain monoamine oxidase in-vivo from the irreversible enzyme inhibition produced by subsequent injection of phenelzine. Levels of protection were similar when the enzyme was assayed with selective substrates (5-hydroxytryptamine and phenethylamine) for both the A and B forms of the enzyme. Although other explanations cannot at this stage be ruled out, these observations are consistent with the tricyclic antidepressants acting as reversible inhibitors of brain monoamine oxidase in-vivo.
Summary. After isolation and purification, rat acute-phase protein, alphamacrofetoprotein and the uterine decidual protein, decidualization-associated protein were compared. They are similar in molecular weight and amino acid composition, and behave similarly during native polyacrylamide gel electrophoresis and are crossreactive with polyclonal antisera. They differ in pI values: with a mean pI of 4·83 for alphamacrofetoprotein, 5·16 for the major form of decidualization-associated protein and 4·97 for the remainder. Alphamacrofetoprotein and decidualization-associated protein differ in carbohydrate content and subunit structure, but show similar susceptibility to trypsin digestion. Evidence is provided that both the decidual species form complexes with proteases and that these are present in the extracted decidual proteins. The distribution of the forms of decidualization-associated protein in the tissues during pregnancy and parturition is described and discussed. Keywords: α-macrofetoprotein; decidual tissue; early pregnancy; decidualization-associated protein; rat
ABSTRACT Improved immunosuppressive regimens, better postoperative intensive care and judicious patient selection have all resulted in increased patient survival following orthotopic liver transplantation (OLT), which has become the preferred option for most patients with end‐stage primary biliary cirrhosis (PBC). As with most other clinical series, PBC is now the most common indication for OLT in the King's College hospital and Cambridge programmes. To date (30 July 1990), 129 patients with PBC have been transplanted, with overall actual 1 and 5 year survival rates of 65 and 63% respectively. When patients transplanted since 1985 are considered, both the 1 and 2 year survival rates are 78%. Immediate operative morality was 4.5%, generally due to uncontrollable bleeding, while further mortality within 30 days of operation—mainly consequent upon infection and multi‐organ failure—has fallen from 40% prior to 1985 to 9% since 1988. Thirteen per cent of patients have been retransplanted for vanishing bile duct syndrome, manifest in this series invariably within the first 6 months following OLT. Although rehabilitation in this series was excellent, a significant percentage of cases have continuing problems with metabolic bone disease, hypertension and renal impairment, mainly due to cyclosporin toxicity.
The aim of this study was to develop an effective and nontoxic vaccine, suitable for use in humans, which was capable of effectively controlling oestrogen levels. Female Sprague-Dawley rats were immunized with a conjugated analogue of gonadotrophin releasing hormone, GnRH-glycys-PPD. This resulted in high levels of neutralizing antibody which disrupted GnRH function and consequently caused a reduction in serum oestrogen. The effect of oestrogen deprivation correlated well with ovarian failure and gonadal atrophy. An examination was made of various adjuvants in conjunction with the analogue to determine the suitability of the combinations in the formulation of an effective human vaccine. This investigation included a novel adjuvant, non-ionic surfactant vesicles (NISV); the results showed that NISV are completely nontoxic and in terms of potentiating and sustaining an immune response, compare favourably with Freund's adjuvant and alum. In addition the long term effects of immunization were investigated and the data showed that immunoneutralization of GnRH effectively suppresses fertility on a long-term basis.
We report a case of histiocytic necrotizing lymphadenitis without granulocytic infiltration (Kikuchi-Fujimoto disease), diagnosed at necropsy in a 19-month-old child dying unexpectedly after a febrile illness. This is the youngest case with this disease that has been thus far reported. It is one of only two reported cases in which the patient died during the acute phase of the illness. Histological findings not unlike those seen in the lymph nodes were present at extranodal sites; this is the first case in which this feature has been described. In keeping with many other reported cases, it was not possible to identify an underlying etiology that might explain the morphologic changes.
ABSTRACT. Cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD‐I mice using standardized protocols and a well‐characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose‐response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose‐response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.
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