An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants.

We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating DNA ploidy using a plate loader on a BD FACS-Canto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone sample handling. In two 96-well plates of samples, the histogram peaks of DNA content from flow cytometry were wellresolved in 189 of 192 samples tested (98.4%), with CV values ranging from 2.98% to 6.20% with an average CV of 4.35% (SD = 0.68%). This new method is useful in doubled haploid plant breeding programs where early discrimination of haploid and doubled haploid (i.e., diploid) plantlets can confer significantly improved operational efficiencies. We discuss how this method could be further refined including adapting the method to robotic sample processing.

An outbreak of multi-resistant Shigella sonnei in a long-stay geriatric nursing centre.

An outbreak of Shigella sonnei infection in a long-stay nursing centre was detected during routine surveillance of notifications in July 1999. Subsequent investigations identified 13 cases of multi-resistant S. sonnei infection affecting nine staff, three community members associated with the centre and one resident of the centre. Each isolate of S. sonnei was genetically indistinguishable. The outbreak investigation identified contact with residents with vomiting and diarrhoea as a significant risk factor for infection amongst staff providing nursing care. This association, and the duration of the outbreak over several months, suggests that transmission was most likely person-to-person. This outbreak demonstrates the importance of infection control policies and hygiene measures in long-stay nursing facilities.

Potential exposure to Australian bat lyssavirus, Queensland, 1996-1999.

Two human deaths caused by Australian bat lyssavirus (ABL) infection have been reported since 1996. Information was obtained from 205 persons (mostly adults from south Brisbane and the South Coast of Queensland), who reported potential ABL exposure to the Brisbane Southside Public Health Unit from November 1,1996, to January 31, 1999. Volunteer animal handlers accounted for 39% of potential exposures, their family members for 12%, professional animal handlers for 14%, community members who intentionally handled bats for 31%, and community members with contacts initiated by bats for 4%. The prevalence of Lyssavirus detected by fluorescent antibody test in 366 sick, injured, or orphaned bats from the area was 6%. Sequelae of exposure, including the requirement for expensive postexposure prophylaxis, may be reduced by educating bat handlers and the public of the risks involved in handling Australian bats.

A revised model of platelet aggregation.

In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates.

Hepatitis A outbreaks among illicit drug users and their contacts in Queensland, 1997.

OBJECTIVE: To describe five outbreaks of hepatitis A virus (HAV) infection associated with illicit drug use during a statewide outbreak of HAV infection in Queensland. DESIGN: Risk factor prevalence survey. PATIENTS AND SETTING: All 875 cases of HAV infection notified to Public Health Units in Queensland in the 12 months to 30 November 1997. MAIN OUTCOME MEASURE: Type and prevalence of illicit drug use. RESULTS: Risk factor assessment was completed for 804 cases (91.9%). We identified five outbreaks of HAV infection linked to illicit drug use. These outbreaks accounted for 24.6% (215/875) of all notified cases and 39% (190/482) of notified cases in the 15-34 years age group. The main type of illicit drug use in four of the five outbreaks was injecting drug use (74%; 118/160), while in the other outbreak it was sharing of smoking implements for marijuana (38%; 21/55). CONCLUSION: Illicit drug use may be an under-recognised risk factor for HAV infection, particularly in young people. Faecal-oral transmission through poor personal hygiene, including sharing of implements for smoking marijuana, is the most probable route of transmission in these drug-linked outbreaks. The role of contaminated drug and needle-sharing remains to be clarified.

The role of glutaminase in the small intestine.

Glutaminase is the enzyme which hydrolyses glutamine, the main respiratory fuel of the intestine, to yield glutamate and ammonia. Glutaminase has a central role in intestinal metabolism: the products of the reaction catalyzed by glutaminase can be transaminated, catabolized to yield energy or used for the biosynthesis of pyrimidine nucleotides. Experimental treatments which deprive the intestine of glutamine induce intestinal atrophy. In this review, attention is paid to the role of glutaminase in intestinal metabolism. Background information on the structure, kinetics and distribution of glutaminase precede a discussion of the metabolism of glutamine within the intestine. In closing, we review the factors known to regulate glutaminase activity and emphasise that the regulation of glutaminase within the intestine is poorly understood.

The effect of minimum luminal nutrition on mucosal cellularity and immunity of the gut.

Many catabolic patients can only consume small volumes of enteral nutrients. The aim of this study was to evaluate markers of cellularity and immunity in the small intestine of rats randomized to receive 6 days of parenteral nutrition, 25% enteral and 75% parenteral nutrition (i.e. minimum luminal nutrition) or enteral nutrition. The same glutamine-enriched solution was used for both parenteral and enteral nutrition. Enteral nutrition was associated with the least amount of jejunal atrophy (P<0.01), with the results from the minimum luminal nutrition group approximating those of the parenteral nutrition group. Parenteral nutrition was associated with the greatest number of CD2+ cells (P< 0.05) and the lowest CD4/CD8 cell ratio (P< 0.01) in the jejunal mucosa. In essence, we failed to demonstrate that there are any appreciable benefits associated with the enteral consumption of 25% of a nutrient load.

The role of enterocytes in gut dysfunction.

Stem cells in the intestinal epithelium give rise to enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Each of these cell lines plays a role in cytoprotection of the intestinal mucosa. In particular, it has been demonstrated that mature enterocytes can act as antigen presenting cells. Parenteral and enteral nutrition are used to nourish critically ill patients. However, these regimens are unfortunately associated with gut atrophy. Glutamine, the preferred intestinal nutrient, reverses this gut atrophy and plays a key role in maintaining the barrier function of the gut. Specific nutrients (putrescine, spermidine, spermine) have been used to modulate intestinal adaption. In addition, ornithine has been shown to act as a regulator of intestinal adaption. In this review, we discuss the relationship between the biology of enterocytes and failure of the gut barrier.

Quantitation of glutaminase mRNA in enterocytes using competitive RT-PCR.

The concentration of mRNA within the intestinal mucosa is usually measured by either Northern blot analysis or semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). However, these methods are limited by a lack of valid internal controls, low sensitivity, and large differences in the concentration of the internal control and target gene. The authors present an alternative method using competitive RT-PCR to measure glutaminase mRNA in isolated enterocytes.

Influence of ischaemia-reperfusion injury on CD44 expression in rat small intestine.

BACKGROUND: CD44 is an adhesion molecule expressed by neutrophils and lymphocytes which is involved in cell-cell and cell-matrix binding. In this study, the effect of ischaemia-reperfusion injury on CD44 messenger RNA (mRNA) and cell surface immunohistochemical expression of CD44 in the rat small intestine was evaluated. METHODS: Wistar rats (n=16) were randomized to either serve as controls (sham surgery) or to be subjected to a standardized ischaemia-reperfusion injury (suprarenal aorta occluded for 1 h followed by 1 h of reperfusion). Standardized segments of jejunum were harvested after ischaemia-reperfusion injury (ischaemic and reperfused samples) to measure the mucosal protein and DNA content, mRNA expression of CD44 and the immunohistochemical expression of CD44. RESULTS: Reperfusion significantly damaged the jejunal mucosa, e.g. mucosal protein content was lower after reperfusion compared with that in the control group (z=-2.31, P=0.02) and the ischaemic samples (z=-2.52, P=001). The expression of cell surface CD44 protein was also significantly decreased after ischaemic injury (z=-1.99, P=0.04); this coincided with a decrease in the amount of cytoplasmic CD44 mRNA within isolated enterocytes (z=-2.31, P=0.02). CONCLUSION: Ischaemia-reperfusion injury decreases the expression of CD44 within the jejunal mucosa. This may contribute to the failure of the gut barrier after such injury.

Ischaemia-reperfusion injury to the intestine.

Ischaemia-reperfusion injury (IRI) is of obvious relevance in situations where there is an interruption of blood supply to the gut, as in vascular surgery, or in the construction of free intestinal grafts. It is now appreciated that IRI also underlies the guy dysfunction that occurs in early shock, sepsis, and trauma. The events that occur during IRI are complex. However, recent advances in cellular biology have started to unravel these underlying processes. The aim of this review is to provide an outline of current knowledge on the mechanisms and consequences of IRI. Initially, IRI appears to be mediated by reactive oxygen metabolites and, at a later stage, by the priming and activation of polymorphonuclear neutrophils (PMN). Ischaemia-reperfusion injury can diminish the barrier function of the gut, and can promote an increase in the leakage of molecules (intestinal permeability) or the passage of microbes across the wall of the bowel (bacterial translocation). Ischaemia-reperfusion injury to the gut can result in the generation of molecules that may also harm distant tissues.

The influence of ischemia/reperfusion injury on the jejunum.

Ischemia/reperfusion injury (IRI) after free tissue transfer of the small intestine results in transmural tissue damage. This study examined the effects of IRI on the jejunum. Wistar rats served either as controls (N=10) or underwent clamping of the infrarenal aorta for 1 hour followed by 1 hour of reperfusion (N=10). Both ischemia and reperfusion reduced the protein and deoxyribonucleic acid content of the jejunal mucosa (p < 0.05). Myeloperoxidase activity in the jejunal mucosa remained relatively low. The expression of leukocyte function-associated antigen 1 and intercellular adhesion molecule 1 (ICAM-1) on the surface of mucosal cells was not altered significantly by the ischemic insult, but was reduced after the period of reperfusion (p < 0.05). This coincided with an increase in messenger ribonucleic acid (mRNA) for ICAM-1 within isolated mucosal cells (p < 0.05). The specific activity of glutaminase in isolated jejunal mucosal cells was diminished after ischemia and reperfusion (p < 0.05), and this was not associated with an appreciable change in glutaminase mRNA expression. These results have identified some molecular mechanisms underlying IRI of the small intestine that are possible candidates for therapeutic intervention.

The pathobiology of peritonitis.

The peritoneum is more than a mechanical covering that allows for the easy gliding of opposed peritoneal surfaces. The peritoneal mesothelial cells facilitate the action of powerful innate immune mechanisms. In addition, the peritoneal-associated lymphoid tissues contain unique cells that may play a crucial role in the localization of intraperitoneal infection. A clearer understanding of the molecular and cellular events underlying peritoneal functions in both the unstimulated and stimulated state will aid future treatment of peritonitis.

Enteral branched-chain amino acids increase the specific activity of jejunal glutaminase and reduce jejunal atrophy.

Branched-chain amino acid (BCAA)-enriched nutrient solutions reduce gut atrophy associated with parenteral nutrition. We hypothesized that this effect was mediated by phosphate-dependent glutaminase. Thirty male Wistar rats (300-350 g) underwent a standardized surgical procedure and were then randomized into three groups to receive 6 days of ad libitum enteral nutrition. The animals were fed a solution of conventional nutrients, a solution of conventional nutrients enriched with 2.0% BCAA or a solution of conventional parenteral nutrients enriched with 2.5% glutamine. When compared with rats fed conventional nutrients, rats fed BCAA and glutamine had less jejunal atrophy (P < 0.05) and a greater specific activity of phosphate-dependent glutaminase in the jejunum (131%; P < 0.05). It is concluded that enteral BCAA reduce atrophy of the jejunum via the generation of glutamine.

Review: Peyer's patches.

The function of Peyer's patches as antigenic sampling sites involves the complex interplay of a variety of mechanisms that aim to recognize luminal antigens, induce an immunological response and decrease the incidence of antigen translocation across the mucosal epithelium. This is achieved by M cells, which facilitate the uptake of luminal antigens, a vascular architecture that promotes the retention of absorbed antigens within the patch interstitium (allowing for maximal antigenic activation of lymphocytes) and the presence of lymphoid follicles that contain antigen-presenting cells and lymphocytes. Lymphocytes encountering antigen in the Peyer's patches proliferate, differentiate into fully mature antigen-specific effector cells and migrate to the mesenteric lymph nodes where they undergo final maturation. The mature lymphocytes then enter the systemic circulation and migrate throughout the other mucosa-associated lymphoid tissues of the body and "home' into the gut via high endothelial venules and gut-associated lymphoid tissue-specific adhesion molecules, providing antigen-specific lymphocytes at sites likely to re-encounter the antigen.

Peritoneal defences and peritoneum-associated lymphoid tissue.

The peritoneum is mainly protected by the innate immune system. This consists of mechanical clearance of the peritoneal cavity, activation of complement, and the actions of polymorphonuclear neutrophils and macrophages. The specific immune system, which is mediated by the activity of lymphocytes, provides a secondary amplification system that may be of great importance for patients with intraperitoneal sepsis. This review provides an overview of the relevant innate immune mechanisms and explores the possible role of peritoneum-associated lymphoid tissue.

Loss of heterozygosity studies in squamous cell carcinomas of the head and neck.

BACKGROUND: Loss of heterozygosity (LOH) studies have been pivotal in identifying tumor suppressor genes involved in the pathogenesis of a number of cancers. In squamous cell carcinomas of the head and neck region (SCCHN), LOH studies using the Southern blot technique are scarce. METHODS: SCCHNs were obtained immediately after surgical resection from 78 patients. Histologic confirmation was made by frozen section and tumors with less than 50% malignant cells were excluded. DNA was digested with restriction enzymes, and after Southern blotting the membranes were hybridized with radio-labeled probes. Chromosome arms analyzed included 1p, 3p, 4p, Sq, 8p, lOp, 11p, 11q, 13q, 17p, 17q, 18q, 21q, and 22q. RESULTS: The average rate LOH was 25% per chromosome arm. Significantly higher rates of LOH were observed for chromosome arms 5q (56%) and 17p (45%). Other investigators have reported high rates of LOH for the H- ras-1 locus, and chromosome arms 11p, 11q, and 13q. However, these results were not confirmed in this study. For patients with stage 1 or 2 tumors, the overall LOH rate was 13%, and for patients with stage 3 or 4 disease the rate was 23%. This difference was statistically significant (p < 0.025). CONCLUSIONS: tumors progress to higher stages, they appear to accumulate an increasing number of genetic abnormalities. Chromosome arms 5q and 17p contain tumor suppressor genes which are likely to be involved in the pathogenesis of SCCHN:

The effect of branched-chain amino acid-enriched parenteral nutrition on gut permeability.

In situations of catabolic stress, the gut becomes atrophic and has a diminished barrier function as evidenced by an increased permeability to a variety of molecules. It is known that the parenteral administration of branched-chain amino acids (BCAA) reduce gut atrophy. The aim of this study was to examine the effect of BCAA-enriched solutions of parenteral nutrients on gut permeability. A secondary aim was to observe the association between gut permeability and variables that have been used to assess jejunal atrophy. Central venous lines were inserted into 30 rats before randomization to receive nutritional support with: (1) a conventional parenteral solution (CPN), (2) A 2.0% BCAA-enriched solution (BCAA), or (3) rat food ad lib (Rat Food). The rats were assessed after 7 d for nutritional status, gut morphology, and gut permeability ratio (ratio of the permeability to 14C raffinose and 3H mannitol). We found that rats in the Rat Food Group lost the least amount of weight, had the least amount of jejunal atrophy, and had better preservation of barrier function as determined by gut permeability. When compared with the CPN Group, the BCAA Group had better preservation of jejunal morphology and protein content (p < 0.05), but a similar gut permeability. A cross-correlation matrix demonstrated a significant negative correlation between permeability to mannitol and mucosal weight, mucosal protein content and mucosal DNA content. Branched-chain amino acid-enriched parenteral nutrition reduced gut atrophy but not the gut permeability associated with parenteral nutrition. In the parenterally nourished rat model, atrophy of the jejunum is associated with increased permeability to small molecules.

Glutamine.

Glutamine is the most abundant free amino acid in the circulation. It is a primary fuel for rapidly dividing cells and plays a key role in the transport of nitrogen between organs. Although glutamine is absent from conventional regimens aimed at nutritional support, glutamine deficiency can occur during periods of metabolic stress; this has led to the reclassification of glutamine as a conditionally essential amino acid. Experiments with various animal models have demonstrated that the provision of glutamine can result in better nitrogen homoeostasis, with conservation of skeletal muscle. There is also considerable evidence that glutamine can enhance the barrier function of the gut. This review concludes by discussing the clinical evidence that supports the inclusion of stable forms of glutamine in solutions of nutrients.

The effect of minimum luminal nutrition on bacterial translocation and atrophy of the jejunum during parenteral nutrition.

In situations of catabolic stress, the gut becomes atrophic and may have diminished barrier function as evidenced by an increase in bacterial translocation. The aim of this study was to examine the effect of minimum luminal nutrition during parenteral nutrition on the extent of jejunal atrophy and rate of bacterial translocation. Central venous lines were inserted into 30 rats before they underwent randomization to receive nutritional support with: (a) conventional parenteral nutrition; (b) conventional parenteral nutrition with 3 g/day of rat food (i.e., minimum luminal nutrition); or (c) rat food ad libitum. The rats were assessed after 10 days for nutritional status, extent of jejunal atrophy, caecal flora, as well as the extent of bacterial translocation to the mesenteric lymph nodes, liver and spleen. Rats in the rat food ad libitum group lost the smallest amount of weight and had the least amount of jejunal atrophy, yet had a similar rate of bacterial translocation as the parenterally nourished groups. When compared with the conventional parenteral nutrition group, the minimum luminal nutrition group had better preservation of the weight of the small bowel and its isolated mucosa (P < 0.01), but had a similar rate of bacterial translocation. Minimum luminal nutrition reduced the extent of atrophy of the gut but did not affect the incidence of bacterial translocation. It is inferred that there is no direct relationship between the extent of mucosal atrophy and incidence of bacterial translocation.