The clinical utility of genetic testing of tissues from pregnancy losses.

OBJECTIVE: To map the current testing being undertaken following pregnancy loss across the UK and to examine the clinical utility in terms of identifying a cause for the loss and in identifying couples at risk of an unbalanced liveborn child. DESIGN: Retrospective audit. SETTING: UK, for the year 2014. POPULATION: An audit of 6465 referrals for genetic testing of tissue samples following pregnancy loss. METHODS: Data were obtained by questionnaire from 15 UK regional genetics laboratories. MAIN OUTCOME MEASURES: Data were analysed with respect to gestational age, the presence of identified fetal anomalies, methodologies used, abnormality rates and the presence of a parental balanced rearrangement. RESULTS: Of 6465 referrals a genetic cause was identified in 22% of cases (before 12 weeks' gestation, in 47%; at 12-24 weeks, in 14%; after 24 weeks, in 6%). In 0.4% of cases a balanced parental rearrangement was identified where there was a risk of an affected liveborn child in a future pregnancy. Eighty percent of genetic imbalances identified were aneuploidy or triploidy and could be identified by quantitative fluorescence polymerase chain reaction alone. There was significant variation across the UK in acceptance criteria, testing strategies and thus level of resolution of testing. CONCLUSIONS: Genetic testing of tissues following pregnancy loss identifies a probable cause of fetal demise in 22% of cases, but it is of low clinical utility in identifying couples at risk of a future unbalanced liveborn child. A comprehensive multidisciplinary review is needed to develop proposals for an affordable and equitable service. TWEETABLE ABSTRACT: UK audit of genetic testing of fetal loss shows variation in access to and resolution of analysis.

Dental health in rural Victoria: the Crossroads Household Survey.

BACKGROUND: The aims of this study were to describe dental health and perceived barriers to dental care in a regional centre and surrounding smaller towns in rural Victoria. METHODS: A stratified, randomized, face-to-face household survey was undertaken to assess levels of edentulism and access to oral health services. A study response rate of 70.3 per cent (6316/9260) was achieved. RESULTS: When compared with those in the regional centre, people living in the shire capitals were more likely to travel greater distances to see a dentist and were less likely to have seen a dentist within the past 12 months. While there was little difference in the edentulous proportion living in shire capitals compared with the regional centre, the level of edentulousness over the entire region was greater than overall Australian estimates. Differences in perceived barriers to care within the region were less than expected. Existing perceived barriers were lack of need, time until available appointments, attitudes of dentists and lack of public dental facilities. Differences in these barriers existed between socio-economic groups. CONCLUSIONS: This study shows that the prevalence of edentulism was higher in the areas studied relative to the Australian population. Significant patient perceived barriers to care exist which may contribute to the problem.

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva.

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.

Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552.

A Lactococcus lactis subsp. lactis strain (DPC5552), which causes the lysis of other lactococcal cultures, was isolated during a screening of raw milk samples for bacteriocin producers. Purification of the bacteriocin produced revealed that production of the lantibiotic, lacticin 481, was associated with the bacteriolytic capability of the strain. However, unlike bacteriocin-induced lysis observed with bacteriocins such as lacticin 3147 and lactococcins A, B, and M (where the target strain is killed), the DPC5552 supernatant gave rise to a situation whereby the target strain continued to grow (albeit at a lower rate) with simultaneous release of the intracellular enzymes lactate dehydrogenase (LDH) and post-proline dipeptidyl aminopeptidase (Pep X). In parallel experiments, 32 AU/ml of the inhibitory activity from L. lactis DPC5552 resulted in a 10- and 6-fold-higher LDH release after 5 h than that with 32 AU/ml of either lacticin 3147 or lactococcin A, B, and M. Laboratory-scale Cheddar cheese-making trials also demonstrated that lacticin 481-producing cultures induced the release of elevated levels of LDH from the starter L. lactis HP, without severely compromising its acid-producing capabilities. These results indicate that lacticin 481-producing strains may provide improved adjuncts for delivering lactococcal intracellular enzymes into the cheese matrix and, thus, improve cheese quality and flavor.

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems.

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.

Detection of sporadic cases of Norwalk-like virus (NLV) and astrovirus infection in a single Irish hospital from 1996 to 1998.

BACKGROUND: 'Norwalk-like viruses' (NLV) and astroviruses are recognised as the most important etiologic agents of viral gastroenteritis, excluding rotaviruses. However, neither of these two groups of viruses is routinely screened for in Irish hospital laboratories. OBJECTIVE: The objective of this study was to examine faeces collected from patients with non-bacterial, non-rotaviral gastroenteritis and examine if NLVs and astroviruses could be identified as the causative agents of the illness. STUDY DESIGN: Faecal specimens were collected from a single Irish hospital from February 1996 to June 1998. Three hundred and sixty samples were tested for the presence of NLVs using newly designed inosine-containing degenerate primers. Two hundred and three faecal specimens from paediatric patients were screened for the presence of astroviruses. RESULTS: the results of the screening study were that 29 (8%) specimens were found to be positive for NLV by reverse transcription polymerase chain reaction (RT-PCR) and 15 (7%) specimens from paediatric patients were found to be positive for astroviruses. Genotyping of the NLV-positive samples determined that four of the isolates were from genotype I (G1) and 25 were G2. The G2 positive specimens were further subtyped by oligonucleotide probing and the majority (n = 21) were found to be subtype P2-B, with four isolates being typed as P2-A. No P1-B isolates were found. CONCLUSIONS: This is the first report of detection of sporadic cases of NLV and astrovirus in Ireland. The results obtained highlight the need for continued surveillance of these viruses and the development of rapid detection systems for use in clinical laboratories.

Efficient method for the detection of microbially-produced antibacterial substances from food systems.

A novel method for the isolation of microbially-derived inhibitory substances from food sources was developed. The method involves an enrichment step coupled to a killing assay which is initially carried out in multiwell plates. The technique has advantages in that large numbers of samples can be tested in parallel. The assay can be completed in less than 60 h and is more sensitive than direct plating due to the enrichment step. This novel screening approach was compared with the standard direct plating approach in an effort to identify the antimicrobial potential of a number of Kefir grains. Kefir grains were incubated in 10% reconstituted skim milk for 20 h at 32 degrees C to enable production of any potential biopreservatives. Following overnight incubation, fermentates were aliquoted into multi-well plates and a known number of indicator cells was added to each well. The fermentates were incubated for a further 20 h and counts were carried out to determine whether a reduction in indicator cell numbers had occurred. A reduction in cell-forming units indicated the presence of an inhibitory substance and these inhibitory fermentates were selected for further investigation. Using the protocol outlined, Kefir fermentates capable of inhibiting Listeria innocua DPC1770 and Escherichia coli O157:H45 were identified.

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria.

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.

Developing applications for lactococcal bacteriocins.

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrated its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.

Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens.

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10(8) midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C. In the case of S. aureus 10, resuspension of 2.5 x 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30 degrees C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.

Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin.

We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.

The effects of queenlessness on the maturation of the honey bee olfactory system.

During the first week of adult life the olfactory system of the honey bee undergoes a critical period of maturation [Masson and Arnold, Organisation and plasticity of the olfactory system of the honeybee, Apis mellifera, in: Menzel and Mercer (Eds.), Neurobiology and Behaviour of Honeybees. Springer-Verlag, Berlin, 1987, pp. 280 295]. This is accompanied by dramatic increases in the volume of the antennal lobes [Winnington et al., Structural plasticity of identified glomeruli in the antennal lobes of the adult worker honey bee. J. Comp. Neurol., 365 (1996) 479-490], centres of the brain that receive direct input from primary olfactory receptor neurons housed in the antennae of the bee. Here, we show that during the first 4-6 days of adult life there is a significant increase in the percentage of bees that respond to a conditioned olfactory stimulus after a single conditioning trial and, furthermore, that the ontogeny of this olfactory learning behaviour is altered significantly if the queen is removed from the colony. The absence of a queen during early adult life also has site-specific effects on the maturation of the antennal lobes of the brain. These results show for the first time that the queen's presence in a colony has a significant impact not only on the behaviour of the adult worker honey bee, but also on the structure of the brain.

Isochromosomes of both short and long arms of chromosome 12 resulting in an additional copy of chromosome 12 in a case of splenic lymphoma with villous lymphocytes.

We report a case of splenic lymphoma with villous lymphocytes showing a karyotype with an isochromosome for both the long arm and the short arm of chromosome 12, i(12)(p10) and i(12)(q10), effectively resulting in trisomy 12. This is, apparently, the first documented case of an additional copy of chromosome 12 resulting from isochromosome formation in a neoplastic disorder.

Programmed cell death during metamorphosis in the blow-fly Calliphora vomitoria.

During metamorphosis, the salivary glands of the blow-fly undergo programmed cell death. Data is presented indicating that this programmed cell death does not in many respects emulate classical apoptosis. The cells are seen to vacuolate and swell rather than condense and shrink. There appears to be a transient enhancement in autophagy and an increase in acid phosphatase activity. This is followed by the characteristic appearance of ribosomal and extracisternal sources of the enzyme leading to autolysis. There appears to be no lysosomal leakage of acid phosphatase. As in apoptosis, the mitochondria persist until the cell fragments. The nucleus, however, does not show the distinct chromatin margination and blebbing that is typical of apoptosis. These changes are compared with necrotic changes induced by experimental anoxia. Overall the results show that a programmed cell death distinct from classical apoptosis is taking place.

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.

Cell death in the salivary glands of metamorphosing Calliphora vomitoria.

The salivary gland cells of Calliphora vomitoria larvae initiate and complete their own destruction in a programmed manner at the onset of metamorphosis. On entering the post-feeding period the larvae come to rest and the polytene salivary gland cells show a significant increase in DNA synthesis followed closely by a surge of mRNA synthesis accompanied by increasing protein production. During this prelude to cell death the new mRNA gives rise to at least 10 new proteins. The first new proteins having a MWt between 30 and 100kD appear by day 8 of the life-cycle and a number persist until the advent of cell death on day 9. Other new proteins appear in a cascade of production during day 8 and in vitro translation of mRNA produced at this time shows a new 55kD protein appearing before cell destruction. Significantly no evidence of DNA degeneration or laddering associated with classical apoptosis was observed, on the contrary considerable DNA synthesis in the form of chromosomal endoduplication or "genomic amplification" was seen; selective gene expression being apparently controlled at translational level. Overall the results strongly suggest a synthetically mediated programmed cell death in the metamorphosing salivary glands of the blow-fly which is distinct from apoptosis.

The primate hippocampal formation: evidence for a time-limited role in memory storage.

Clinical and experimental studies have shown that the hippocampal formation and related structures in the medial temporal lobe are important for learning and memory. Retrograde amnesia was studied prospectively in monkeys to understand the contribution of the hippocampal formation to memory function. Monkeys learned to discriminate 100 pairs of objects beginning 16, 12, 8, 4, and 2 weeks before the hippocampal formation was removed (20 different pairs at each time period). Two weeks after surgery, memory was assessed by presenting each of the 100 object pairs again for a single-choice trial. Normal monkeys exhibited forgetting; that is, they remembered recently learned objects better than objects learned many weeks earlier. Monkeys with hippocampal damage were severely impaired at remembering recently learned objects. In addition, they remembered objects learned long ago as well as normal monkeys did and significantly better than they remembered objects learned recently. These results show that the hippocampal formation is required for memory storage for only a limited period of time after learning. As time passes, its role in memory diminishes, and a more permanent memory gradually develops independently of the hippocampal formation, probably in neocortex.

Comparative neuropsychology and Korsakoff's syndrome. III--Delayed response, delayed alternation and DRL performance.

Performance of alcoholic Korsakoff patients was compared with that of patients with Huntington's disease. Broca's aphasia or alcoholism (without clinical signs of memory impairment) on delayed alternation (DA) and delayed response (DR) tests. Korsakoffs were impaired on both tasks, and Huntington patients were impaired on DA only. In a separate experiment, performance by Korsakoffs was compared to that of alcoholic and normal controls on four DRL schedules. Korsakoffs tended to be overresponsive, making errors of commission early within a schedule, and consequently, obtaining fewer reinforcements than the controls.