Incidence and pattern of acute anterior uveitis in Central Australia.

AIM: To investigate the incidence and patterns of acute anterior uveitis (AAU) in Central Australia and specifically to study the relative frequency of AAU in Australian Aborigines compared with that in non-Aboriginal patients. METHODS: Prospective, observational study of all patients seen by the Central Australian Ophthalmology service over an 8-month period. RESULTS: The incidence rate of AAU in Central Australia was 35.9 cases/100,000 population per year. Forty-two per cent of the 1955 patients seen during the study period were Australian Aboriginal patients; however, all but one patient with AAU were Caucasian. The difference in the incidence of AAU between the Indigenous and non-Indigenous populations was statistically significant (p = 0.03, Fisher's exact test). Four of the nine Caucasian patients with AAU were HLA-B27-positive. The single case of AAU in an Australian Aborigine was a recurrent episode of HLA-B27-positive AAU. A family history of this patient revealed that both her grandfathers were Caucasian. CONCLUSION: The incidence and pattern of AAU in Central Australia is comparable with that in other geographical regions. However, AAU occurs very infrequently in Australian Aborigines compared with that in the non-Indigenous population of Central Australia, further implicating the importance of genetic factors in the pathogenesis of AAU.

Identifying the origin of single corneal cells by DNA fingerprinting: part I--implications for corneal limbal allografting.

PURPOSE: To demonstrate that the combination of impression cytology and single cell DNA fingerprinting represents a powerful tool that is suitable for detecting transplanted cells after corneal limbal allografting. METHODS: Fifty single cells were obtained by corneal impression cytology from 12 patients undergoing cataract surgery. Individual cells were isolated from samples by micromanipulation. Polymerase chain reaction and short tandem repeat profiling was used to obtain forensic standard "DNA fingerprints" from single cells. Blood samples taken at the time of impression cytology provided control "fingerprints." RESULTS: Informative DNA fingerprints were obtained from all corneal samples and 66% (33 of 50 cells) of isolated single cells. Of all fingerprints obtained, most (91%, 30 of 33 fingerprints) corneal fingerprints matched corresponding blood sample fingerprints. At least one corneal fingerprint matched the corresponding blood sample fingerprint in 83% (10 of 12 patients) of the patients in the study. CONCLUSIONS: This extremely specific single cell DNA fingerprinting system permits accurate identification of individual corneal epithelial cells, allowing very reliable determination of their origin, which will enable host and donor cells to be distinguished from each other after keratolimbal allografting procedures, even if the host and donor are the same sex or siblings. These DNA fingerprinting methods allow assessment of quality and quantity of donor cell survival, as well as survival time. The extreme sensitivity and accuracy of the technique means that should contamination occur, it would be identified, thus ensuring meaningful results.

Identifying the origin of single corneal cells by DNA fingerprinting: part II-- application to limbal allografting.

PURPOSE: Successful limbal allotransplantation allows regression of limbal stem cell deficiency features. Transplant survival is presumed if clinical improvement occurs. However, positive proof of surviving transplanted stem cells remains difficult. This follow-up study attempted to prove donor cell survival 5 years after limbal stem cell allograft in one woman with aniridia. METHODS: Impression cytology and single-cell DNA fingerprinting were used to investigate a previously studied patient. Corneal epithelial cells were harvested from five sites and isolated by micromanipulation. Polymerase chain reaction and short tandem repeat profiling were used to obtain forensic standard "DNA fingerprints" from single cells. (The technique is described in the preceding article, Part I.) Blood samples yielded host and donor DNA for comparison. Negative controls were performed for impression cytology and polymerase chain reaction. Simultaneous micro-scrape samples were also taken. RESULTS: Impression cytology samples permitted informative DNA fingerprints from all corneal sites and represented 76% (23/30) of tested cells. Fifty percent (15/30) of the fingerprints were "specific" but 83% (19/23) matched the host DNA fingerprint. The remaining 17% (4/23) represented contamination from various sources. Specific fingerprints were obtained in 55% (10/18) of the cells from micro-scrape samples. All samples giving sufficient information matched the host DNA fingerprint. All tested blood samples gave specific fingerprints. None of the sampled corneal cells gave a donor DNA fingerprint. CONCLUSIONS: In a single patient, no detectable long-term donor cell survival exists at 5 years. Positive identification would have provided unequivocal proof of donor cell survival. This technique gives useful information even if contamination occurs.

The long term outcome of limbal allografts: the search for surviving cells.

BACKGROUND/AIMS: Limbal allotransplantation is increasingly being used for ocular surface repair in patients with limbal stem cell dysfunction. However, it is uncertain whether donor cells survive long term on the ocular surface and whether patients maintain the early benefits of the procedure. The aims of this study were to investigate the long term outcome of clinical limbal allografts and to correlate outcome with donor cell survival. METHODS: Five patients who had undergone allotransplantation-four keratolimbal allografts and one tarsoconjunctival allograft-from 3-5 years previously, and for whom residual frozen donor ocular tissue was available, were reviewed. Survival of donor cells lifted from the recipient ocular surface by impression cytology was investigated by DNA fingerprinting using primers detecting variable nucleotide tandem repeat sequences. Recipient buccal cells and scleral samples from the remnant donor eye were used to genotype recipients and donors, respectively. Polymerase chain reaction products were sized by Genescan analysis. RESULTS: An objective long term benefit from the procedure (improved Snellen acuity, reduced frequency of epithelial defects, reduced vascularisation, and scarring) was recorded for four patients. Some subjective benefit was also reported. However, in no instances were donor cells recovered from the ocular surface at 3-5 years post-graft. Initial experiments to examine sensitivity indicated that any surviving donor cells must have constituted less than 2.5% of cells sampled. CONCLUSION: Limbal stem cell allotransplantation can provide long term benefits, as measured by objective criteria. However, such benefits do not necessarily correlate with survival of measurable numbers of donor cells on the ocular surface.

Long-chain polyunsaturated fatty acids (LC-PUFA) during early development: contribution of milk LC-PUFA to accretion rates varies among organs.

Long-chain polyunsaturated fatty acids (LC-PUFA) accretion (essential for growth and neural development) was studied from late fetal throughout weaning age in the ferret, a species with maternal LC-PUFA sufficiency during pregnancy and lactation. The data show that a) accretion rate of LC-PUFA is rapid during early postnatal development, b) milk LC-PUFA decrease during lactation, c) adipose tissue LC-PUFA level is directly related to milk LC-PUFA level, while accretion in brain and liver exceeds dietary intake, d) accretion of arachidonic acid occurs earlier than docosahexaenoic acid, suggesting earlier development of n6-fatty acid endogenous synthesis.

Gastric proteolysis in preterm infants fed mother's milk or formula.

Gastric proteolysis is assumed to be low in the newborn (Britton & Koldovsky 1989). Postprandial pepsin output is significantly lower in preterm infants than adults, 589 vs. 3352U/kg, respectively (Armand et al. 1995, 1996). We now report on gastric proteolysis in preterm infants (gestation age, 29 weeks; postnatal age, 5-6 weeks) gavage-fed mother's milk or preemie formula. The data show that a) the nonprotein component is higher in human milk than formula, b) net proteolysis amounts to 15% of protein, c) gastric proteolysis is lower than lipolysis and, contrary to the latter, is not enhanced by milk feeding (Armand et al. 1996). We suggest that stomach pH, enzyme output, and food structure are the reasons for differences in gastric digestion of protein and fat in infants.

Ascertainment of birth defects: the effect on completeness of adding a new source of data.

BACKGROUND: The Western Australian (WA) Birth Defects Registry aims for complete ascertainment of birth defects in WA, but the proportions of birth defects in rural areas and in Aboriginal children are lower than in metropolitan and non-Aboriginal children. The effect on ascertainment of adding data from the Rural Paediatric Service (RPS) was investigated. METHOD: A file of all cases of birth defects for children born 1980-1997 and recorded on the RPS database was linked to the Registry. RESULTS: The addition of this new data source had little effect on the overall prevalence of birth defects (an increase from 5.38 to 5.41%). There was a slightly greater effect on the prevalence of birth defects in rural residents (4.67%-4.76%) and Aboriginal children (4.55-4.78%), although the prevalence for each of these groups is still less than for metropolitan residents and non-Aboriginal infants, respectively. All major categories of birth defects were represented in the new cases and, in general, their addition made little difference to the prevalence of each category. The exception was fetal alcohol syndrome, which increased from 0.13 per 1000 to 0.18 per 1000 once the 21 new cases from the RPS were added. CONCLUSION: Complete ascertainment of birth defects is important in developing and evaluating preventive programs, and in investigating clusters of birth defects.

Current Australian practice in the prevention and management of corneal allograft rejection.

PURPOSE: To determine current practices in the prevention and management of corneal allograft rejection in Australia. METHODS: A questionnaire was circulated to attendees at the 1998 Eye Bank Meeting in Adelaide. Twenty-four responses were received and analysed. RESULTS: All respondents used topical corticosteroids for routine prophylaxis and to treat established rejection episodes. Prednisolone acetate was the most frequently prescribed topical corticosteroid. Systemic non-steroidal immunosuppression was prescribed almost exclusively for high-risk grafts. Seventy-five per cent of surgeons used systemic antiviral agents for the treatment of graft rejection in patients with Herpes simplex keratitis. CONCLUSION: There was a wide variation amongst surgeons in the choice of therapy for routine prophylactic immunosuppression as well as for the treatment of established corneal allograft rejection.

Protective function of human milk: the milk fat globule.

Human milk contains many components that protect the newborn against infection at a time when the infant's own defense mechanisms are poorly developed. Fat is one of the major nutrients in human milk. The fat is contained within milk fat globules composed of a core of triglyceride and a membrane consisting of phospholipids, cholesterol, proteins, and glycoproteins. Both the membrane and the core components can provide protection against microorganisms. The major protective membrane glycoproteins, mucin, and lactadherin are resistant to conditions in the newborn's stomach and maintain their structure and function even at low pH and in the presence of the proteolytic enzyme pepsin. The core triglycerides upon hydrolysis by digestive lipases (especially gastric lipase, which is well developed in the newborn) produce free fatty acids and monoglycerides, amphiphylic substances able to lyse enveloped viruses, bacteria, and protozoa. Therefore, in addition to its nutritional value, the fat in human milk has a major protective function.

Milk fat globule glycoproteins in human milk and in gastric aspirates of mother's milk-fed preterm infants.

Human milk fat globule (HMFG) glycoproteins can prevent infections by microorganisms in breast-fed infants; the MUC-1 mucin inhibits binding of S-fimbriated Escherichia coli to buccal mucosa, and lactadherin may prevent symptomatic rotavirus infections. In this study, the survival of these HMFG glycoproteins in the stomach of human milk-fed preterm infants (gestational age = 27.5 +/- 0.4 wk) was assessed, and levels in their mothers' milk determined, using specific RIAs. Butyrophilin, a major component of HMFG membrane that has no demonstrated antimicrobial activity, was studied for comparison. The levels of mucin, lactadherin, and butyrophilin in 41 milk samples of 20 mothers were 729 +/- 75, 93 +/- 10, and 41 +/- 3 microg/mL, respectively. Mucin and lactadherin were significantly higher in early milk samples (<15 d postpartum) than in later milk samples (15-90 d postpartum), whereas butyrophilin showed no such difference. Significant amounts of mucin and lactadherin were found in almost all gastric aspirates of human milk-fed infants, even 4 h after feeding (mucin, 270 +/- 30 microg/mL; lactadherin, 23.2 +/- 4.4 microg/mL), whereas butyrophilin was rapidly degraded in the majority of aspirates. Western blot analysis demonstrated that the immunoreactive mucin, lactadherin, and butyrophilin in the milk-fed gastric aspirates had the expected native molecular weights. Mucin and lactadherin survived at all gastric pH values, whereas butyrophilin was found only at pH > 4. Neither lactadherin nor butyrophilin were detected in gastric aspirates of formula-fed infants (gestational age = 27.8 +/- 0.5 wk), whereas the very low level of mucin (9.1 +/- 1.1 microg/mL) in this group is presumably cross-reacting gastric mucin. These results demonstrate that two HMFG glycoproteins implicated in prevention of infection, MUC-1 mucin and lactadherin, survive and maintain their integrity in the stomachs of human milk-fed preterm infants.

Effect of pasteurization on long chain polyunsaturated fatty acid levels and enzyme activities of human milk.

Milk fatty acids, including the polyunsaturated long chain fatty acids essential for retinal function and brain development, are not affected by pasteurization (62.5 degrees C for 30 min). Milk lipases are completely destroyed by pasteurization, whereas amylase lost only 15% of initial activity. Thus, certain bioactive components are stable to pasteurization of donor milk and can benefit the recipient infants.

Gastric lipase and pepsin activities in the developing ferret: nonparallel development of the two gastric digestive enzymes.

BACKGROUND: Gastric lipase has an important compensatory function in neonatal fat digestion. The activity level of pepsin and its role in protein digestion is less well understood. We have, therefore, studied the ontogeny of lipase and pepsin in the ferret, a species with a neonatal fat digestion pattern similar to that of humans. METHODS: Gastric lipase and pepsin activities were quantified from the late fetal period throughout lactation, and were compared with those of the adult. RESULTS: The data show earlier ontogeny and much more rapid rise of lipase activity than of pepsin. Lipase activity was present during the last week of fetal development, whereas pepsin was detected only postnatally. Lipase activity was 72.8% +/- 14.2% and 153% +/- 9.95% and pepsin activity was 11.6% +/- 1.3% and 30.1% +/- 1.3% of the adult level at 2 and 4 wk of age, respectively. CONCLUSIONS: We conclude that lipase activity develops early and exceeds adult activity during the suckling period, when fat intake is very high. The low pepsin activity and high postprandial pH probably limit gastric proteolysis, thereby contributing to the structural and functional stability of milk proteins, many with protective or bioactive function in the gastrointestinal tract of the newborn.

Digestive enzymes in human milk: stability at suboptimal storage temperatures.

BACKGROUND: Women who return to work outside of the home while still breastfeeding must often store the expressed milk at less than optimal temperatures. Human milk provides digestive enzymes (amylase and lipase) that compensate in the newborn for immature pancreatic function. METHODS: We have assessed the stability of amylase and bile salt-dependent lipase after storage for 1-24 h at 15, 25, and 38 degrees C. RESULTS: Both enzymes were stable at 15 and 25 degrees C for 24 h, whereas at 38 degrees C there was a 15 and 20% decrease in lipase and amylase activity, respectively. The stability of milk lipoprotein lipase was also tested. This very labile enzyme was more stable in milk than previously reported for blood and tissues, i.e., 20 and 50% decrease in activity after storage at 15 or 25 degrees C for 24 h, respectively. A two-unit drop in milk pH by 24 h of storage would not affect the activity of digestive enzymes, which are stable at pH > 3.5. CONCLUSIONS: We conclude that milk provides the same compensatory digestive activity after short-term storage, even at relatively high temperature, as when fed fresh to the infant.

Do transplanted corneal limbal stem cells survive in vivo long-term? Possible techniques to detect donor cell survival by polymerase chain reaction with the amelogenin gene and Y-specific probes.

PURPOSE: To investigate donor cell survival following corneal limbal stem cell grafting, which is based on the corneal stem cell model. METHODS: We describe the use of the amelogenin gene probe with the polymerase chain reaction (PCR) to detect surviving donor cells and report preliminary studies using Y-specific DNA probes. RESULTS AND CONCLUSIONS: DNA polymorphisms have a detection limit of 10%. The SRY 'Y-specific' probe has a theoretical detection limit of 1 cell in 10,000. The techniques were applied to investigate survival of male donor cells in an aniridic female patient 2 1/2 years following limbal stem cell grafts. We speculate that low levels of donor-derived cells may still be present. We discuss the advantages and disadvantages of the two approaches, which may have future clinical and experimental application.

Effect of human milk or formula on gastric function and fat digestion in the premature infant.

The effect of diet, human milk or formula, on gastric function (lipase and pepsin activity, pH, and volume) and intragastric digestion of fat was assessed in 28 appropriate for gestational age preterm infants (gestational age, 28.9 +/- 1.4, 29.1 +/- 0.9, 29.5 +/- 0.6 wk; birth weight, 1.00 +/- 0.14 to 1.18 +/- 0.07 kg). The infants were fed either human milk (n = 11), SMA Super Preemie formula (n = 9), or Similac, Special Care formula (n = 8). Fasting and postprandial activity of digestive enzymes, pH, and gastric volume (measured before or during 50 min after gavage feeding) did not differ as a function of diet among the three groups of infants. Gastric lipase output, 23.1 +/- 5.1, 28.3 +/- 6.6, and 22.5 +/- 6.4 (U/kg of body weight) in human milk-, SMA SP-, or Similac SC-fed infants was comparable to the gastric lipase output of healthy adults fed a high fat diet (22.6 +/- 3.0). Pepsin output was, however, significantly lower (597 +/- 77, 743 +/- 97, and 639 +/- 142 U/kg of body weight) in human milk-, SMA SP-, and Similac SC-fed infants) than in healthy adults (3352 +/- 753 U/kg). The hydrolysis of dietary fat was 1.7-2.5-fold higher (p < 0.01) in human milk-fed infants than in infants fed either formula. We conclude that differences in type of feeding, i.e. different fatty acid profiles (long chain or medium chain triglycerides), different emulsions (natural or artificial), and different fat particle sizes do not affect the level of activity of gastric enzymes. However, the triglyceride within milk fat globules appears to be more accessible to gastric lipase than that within formula fat particles. We suggest that the contribution of gastric lipase to overall fat digestion might be greater in the newborn (a period of pancreatic insufficiency) than in the adult.

Digestive lipases of the newborn ferret: compensatory role of milk bile salt-dependent lipase.

The amount of mRNA hybridizing to bile salt-dependent lipase and to colipase-dependent lipase probes as well as their translation into active proteins were quantified in the adult and newborn pancreas and lactating mammary gland from the ferret, a species whose milk, similar to that of the human, has bile salt-dependent lipase. The concentration of colipase-dependent lipase mRNA correlated with the amount of activity found in the adult and newborn pancreas, whereas neither mRNA nor activity of this enzyme was detected in the kit pancreas or in the lactating mammary gland. These data indicate that colipase-dependent lipase is actually expressed in adult pancreas and might represent the main lipolytic system in the adult. mRNA hybridizing to the bile salt-dependent lipase probe used in this study were detected in adult and in newborn ferret pancreas as well as in lactating mammary gland. However, the bile salt-dependent lipase activity expressed in the newborn pancreas was very low when compared with the activity expressed either in the mammary gland or in the adult pancreas. These data argue for a compensatory role of milk bile salt-dependent lipase in lipid digestion in the newborn. The hydrolysis of dietary fat might be initiated by preduodenal lipase, the activity of which is only two times lower in the gastric mucosa of the newborn than in the adult ferret. The high concentration of mRNA hybridizing to the bile salt-dependent lipase probe associated with a very poor bile salt-dependent lipase activity and protein suggests either that these mRNA are very unstable or that they are poorly translated into an active pancreatic bile salt-dependent lipase.

Breastfeeding and the working mother: effect of time and temperature of short-term storage on proteolysis, lipolysis, and bacterial growth in milk.

BACKGROUND: Women who breastfeed have to store expressed milk while at work for later feeding to their infants; however, storage conditions are often not optimal. OBJECTIVE: Top assess microbial growth and stability of milk protein and lipid at 15 degrees C to 38 degrees C for up to 24 hours. METHODS: Sixteen healthy women who breastfed exclusively, either at home (n=11) or who expressed milk for their infants (n=5), were studied during early (1 month) or late (5 to 6 months) lactation. Expressed milk was stored at 15 degrees C, 25 degrees C, and 38 degrees C for 1 to 24 hours for quantitation of pH, proteolysis, and lipolysis; bacterial growth was quantified at 0, 4, 8, and 24 hours of storage. RESULTS: Milk pH decreased 2 units by 24 hours of storage at all temperatures tested. Proteolysis was minimal during milk storage at 15 degrees C or at 25 degrees C for 24 hours and was apparent only after 24 hours of storage at 38 degrees C. Lipolysis was rapid, starting in the first hours of storage and progressing to 8% at 24 hours. Thus, while the greatest increment in proteolysis products was a 40% increase above baseline after 24 hours of storage at 38 degrees C, free fatty acid concentration at this storage time was 440% to 710% higher than in freshly expressed milk. Bacterial growth was restricted mainly to nonpathogens, was minimal at 15 degrees C throughout the 24 hours of storage, was low at 25 degrees C for the first 4 to 8 hours, and was considerably higher at 38 degrees C even during the relatively short period of 4 hours. CONCLUSIONS: Storage of human milk is safe at 15 degrees C for 24 hours, whereas at 25 degrees C it is safe for 4 hours. Milk should not be stored at 38 degrees C. Minimal proteolysis during storage suggests that milk proteins probably maintain their structure and function during short-term storage, while the marked lipolysis might slow bacterial growth during this time.

Peribulbar anaesthesia for cataract surgery: prilocaine versus lignocaine and bupivacaine.

PURPOSE: Prilocaine has recently been introduced for use in ocular local anaesthesia. A prospective randomised double-masked study was undertaken to assess the efficacy of prilocaine 2% plain versus a mixture of lignocaine 1% and bupivacaine 0.5%, each with hyaluronidase. METHODS: Seventy-five patients were recruited. Local anaesthetic was given by a two-injection transconjunctival peribulbar technique. Injection and perioperative pain were graded by visual analogue pain score (range 0-10). Akinesia and orbicularis function were graded by the surgeon. RESULTS: The two anaesthetic mixtures were comparable in efficacy in producing anaesthesia and akinesia. Using the Mann-Whitney U-test for significance, pain of injection ranked as a mean of 0.88 for prilocaine and 1.03 for lignocaine and bupivacaine (p = 0.48, U = 635.5) Perioperative pain was ranked as a mean of 1.17 for prilocaine and 0.91 for lignocaine and bupivacaine (p = 0.41, U = 629.0). CONCLUSIONS: Prilocaine is a useful alternative anaesthetic agent for eye surgery that has low toxicity and is effective without adrenaline.

Plasma lecithin: cholesterol acyltransferase and plasma lipolytic activity in preterm infants given total parenteral nutrition with 10% or 20% Intralipid.

The effect of 10% or 20% Intralipid on lipid clearing enzymes, plasma lipids and apoproteins was investigated during the first 5 days after birth in 37 premature infants maintained on total parenteral nutrition; 21 infants received 20% and 16 received 10% Intralipid, respectively. Lipid was infused over a 20-h period at rates of 1, 2 and 3 g/kg/day on consecutive days. Plasma lecithin: cholesterol acyltransferase (LCAT) activity was low and increased significantly (p<0.05) only during infusions of 3 g/kg/day in both groups of infants. Plasma lipolytic activity was generally not affected by the regimen or preparation (10% or 20%) of Intralipid infused, except for higher (p<0.05) levels at 3 g/kg/day of 20% compared with prelipid infusion. Plasma triglyceride concentrations wer similar after 10% or 20% Intralipid, whereas plasma total cholesterol was significantly higher during infusion of 2 and 3 g/kg/day of 10% compared with 20% Intralipid. The efficient clearing of 20% Intralipid might be related to the lower lecithin: triglyceride ration which is compatible with the low LCAT activity of premature infants.