Increased peripheral blood CD4+ T cell responses to deamidated but not to native gliadin in children with coeliac disease.

T cell recognition of gliadin from dietary gluten is essential for the pathogenesis of coeliac disease (CD). The aim of the present study was to analyse whether gliadin-specific T cells are detectable in the circulation of children with newly diagnosed coeliac disease by using a sensitive carboxfluorescein diacetate succinimidyl ester (CFSE) dilution method. Peripheral blood CD4(+) T cell responses were analysed in 20 children at diagnosis of CD and compared to those in 64 healthy control children carrying the CD-associated human leucocyte antigen (HLA)-DQ2 or -DQ8 alleles. Deamidated gliadin (gTG)-specific T cells were detectable in the peripheral blood of more than half the children with CD (11 of 20, 55%) compared to 15 of 64 (23.4%) of the control children (P = 0.008). Proliferative responses to gTG were also significantly stronger in children with CD than in controls (P = 0.01). In contrast, T cells specific to native gliadin were detectable at comparable frequencies in children with CD (two of 19, 10.5%) and controls (13 of 64, 20.3%). gTG-specific T cells had a memory phenotype more often than those specific to native gliadin in children with CD (P = 0.02), whereas controls had similar percentages of memory cells in both stimulations. Finally, gTG-specific CD4(+) T cells had a higher expression of the gut-homing molecule ß7 integrin than those specific to the control antigen tetanus toxoid. Collectively, our current results demonstrate that the frequency of circulating memory CD4(+) T cells specific to gTG but not native gliadin is increased in children with newly diagnosed CD.

Iron status and haematological changes in adolescent female inpatients with anorexia nervosa.

OBJECTIVES: (i) To investigate the incidence of iron deficiency (both latent iron deficiency and iron deficiency anaemia) in post menarchal female adolescent patients hospitalized with anorexia nervosa. (ii) To observe changes in iron status during refeeding. METHODS: During the study period all post menarchal female patients admitted to the adolescent unit with anorexia nervosa were invited to participate. Ferritin, serum iron, transferrin and transferrin saturation were measured on admission and discharge. Haemoglobin, haematocrit, weight, and body mass index were monitored weekly. RESULTS: All 12 eligible patients participated in the study. Four patients had elevated ferritin levels on admission. Ten of the 12 showed a statistically significant fall in serum ferritin at time of discharge compared with admission (P = 0.004). One of 12 patients had a low serum iron at presentation. All 12 had normal serum iron levels at discharge. Nine of the 12 subjects had a higher serum iron at discharge which was statistically significant (P = 0.046). There was no significant change in haemoglobin levels when comparing admission and discharge levels. Nine of the 12 patients however, transiently developed anaemia, at some stage during treatment. CONCLUSIONS: Iron deficiency is uncommon at both presentation and after nutritional rehabilitation in post menarchal adolescent females with anorexia nervosa. This is most likely due to increased iron storage secondary to the contraction of the circulating blood volume and reduced iron losses from (secondary) amenorrhoea. The fall in ferritin observed during treatment is due to haemopoiesis necessary to fill the increased blood volume associated with weight gain. This also results in normalization of the haemoglobin and haematocrit.

Severe cyclical thrombocytopenia in a patient with a large lymphatic-venous malformation: a potential association?

The case is reported of an infant who had a large vascular malformation involving his left arm and axilla. It was initially believed to be purely lymphatic in composition but some venous elements were identified subsequently, at operation. The lesion was unusual in that there was a total absence of skin over one area of it at birth, that it underwent spontaneous shrinkage in the early weeks of life, and that a circumferential scarring developed which led to severe functional disability of the limb. At 12 months of age the patient developed a profound cyclic thrombocytopenia that spontaneously resolved after 1 year. The cause of the platelet cycling is unresolved but might have been secondary to intermittent production by the malformation of a cytokine which was destructive against the platelets.

Iron deficiency in Australian-born children of Arabic background in central Sydney.

OBJECTIVES: To determine the prevalence of iron depletion and deficiency, and iron-deficiency anaemia, along with risk factors for iron depletion, in Australian-born children aged 12-36 months of Arabic-speaking background. DESIGN: Community-based survey. SETTING: Central Sydney Area Health Service (CSAHS), NSW, April to August, 1997. PARTICIPANTS: All children born at five Sydney hospitals between 1 May 1994 and 30 April 1996, whose mothers gave an Arabic-speaking country of birth and resided in the area served by the CSAHS. MAIN OUTCOME MEASURES: Full blood count (haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume), plasma ferritin concentration, haemoglobin electrophoresis, potential risk factors for iron depletion. RESULTS: Families of 641 of the 1,161 eligible children were able to be contacted, and 403 agreed to testing (response rate, 62.9% among those contacted). Overall, 6% of children had iron-deficiency anaemia, another 9% were iron deficient without anaemia, and 23% were iron depleted. Multiple logistic regression analysis showed three significant independent risk factors for iron depletion: <37 weeks' gestation (odds ratio [OR], 5.88, P=0.001); mother resident in Australia for less than the median time of 8.5 years (OR, 1.96, P=0.016); and daily intake of >600 mL cows' milk (OR, 3.89, P=<0.001). CONCLUSION: Impaired iron status is common among children of Arabic background, and targeted screening is recommended for this group.

Karyotype 69,XXX/47,XX,+15 in a 2 1/2 year old child.

We present the clinical findings in a 2 1/2 year old girl with an unusual mosaic karyotype. Amniocentesis was performed at 35 weeks because of intrauterine growth retardation. The in situ cultures showed 47,XX,+15 in seven colonies, 69,XXX in four colonies, and in two colonies 46,XX was detected. Subcultures showed 69,XXX/47,XX,+15 with no normal cells. A small dysmorphic baby was born at term. Cytogenetic studies were performed on cord blood, amnion, and placental tissue immediately after birth and further studies on peripheral blood, bone marrow, muscle biopsy, and skin cultures at 1 1/2 years of age. FISH with two autosomal centromeric probes was performed on the peripheral blood sample. A normal cell line could not be seen in any postnatal tissue by either technique. The predominant cell line postnatally was 69,XXX. There were no cytogenetic polymorphisms and the parental origin of the different cell lines was not determined. Marked red cell macrocytosis of peripheral blood was noted on routine blood count. Bone marrow aspiration showed megaloblastic haemopoiesis without evidence of vitamin B12 or folate deficiency. At 2 1/2 years, the patient has significant developmental problems.

Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand.

BACKGROUND: Inhibitory antibodies which neutralise factor VIII develop in 10-20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity. AIMS: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22. METHODS: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A. RESULTS: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients, 1-19% in six, 20-39% in 11 and 40-80% in five. In six of nine patients with acquired haemophilia cross-reactivity was < or = 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor. CONCLUSIONS: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor VIIa or prothrombin complex concentrates.

Iron status and anaemia in preschool children in Sydney.

The purpose of this study was to determine the iron status of preschool children in Sydney. We assessed 678 children aged 9 to 62 months living in 32 randomly selected census collection districts in central and southern Sydney for iron status using plasma ferritin; of these 678 children, 542 had zinc protoporphyrin tests, red cell indices and haemoglobin tests. Risk factors for iron deficiency were assessed by an administered questionnaire. Overall, the prevalence of iron depletion was 10.5 per cent, iron deficiency 2.8 per cent and iron deficiency anaemia 1.1 per cent. The 24-to-35-month age group (176 children) had the highest prevalence of iron deficiency anaemia of 3.0 per cent although iron depletion (18.7 per cent) and iron deficiency (5.4 per cent) were highest among the 9-to-23-month age group (182 children). Low iron status was related to age of under 24 months (odds ratio (OR) 2.86,95 per cent confidence interval (CI) 1.72 to 4.76). After adjustment for this age effect, the consumption of red meat fewer than four times a week was significantly associated with iron depletion (OR 2.27, CI 1.25 to 4.17) and there was a tendency for children who were being given a vitamin supplement to be less likely to be iron depleted (OR 4.00, CI 0.95 to 16.67). Iron deficiency and iron deficiency anaemia do not represent a major public health problem in preschool children in Sydney. However, for children in the age range of 12 to 36 months there is scope for interventions to further reduce the prevalence of iron deficiency anaemia.

The 1591C mutation in triosephosphate isomerase (TPI) deficiency. Tightly linked polymorphisms and a common haplotype in all known families.

In order to investigate the basis of the repeated occurrence of the 1591C mutation (TPI 1591C, 105 Glu-Asp) in multiple unrelated families throughout the world, we studied five microsatellite and short tandem repeat markers that lie within a 1.77 megabase region which includes the TPI gene. We also studied an intragenic polymorphic marker that lies within intron 5 of the TPI gene. This polymorphism, recently described by others, is characterized by either an A or a G at position 2262 (the A in the initiation ATG is designated as +1 for both genomic and cDNA nucleotides). With very minor exceptions, all of the known families in the world with the 1591C mutation were available for study. These included five families from the U.S., three from France, one from Greece, one (of Turkish origin) from Germany, and two from Australia. Although we did not have the opportunity to directly study five families from the U.K., key data concerning the 2262 intragenic polymorphism in these subjects were made available to us. Four of the microsatellite and short tandem repeat markers were linked, but in apparent equilibrium. In contrast, a polymorphic repeat pentamer in the CD4 gene, thought to lie telomeric to TPI, was in apparent complete linkage disequilibrium with the TPI 1591C mutation. The intragenic polymorphism was also in apparent complete linkage disequilibrium with the mutation. In unrelated persons of known phase (1591C homozygotes or normal controls), the comparative allele frequencies for the CD4 pentameric repeat were 1.0 (14/14 alleles) in homozygous TPI 1591C subjects and 0.412 (28/68 alleles) in normal subjects (p < 0.0001). Again, in persons of known phase, the comparative allele frequencies for the A form of the intragenic 2262 A or G polymorphism were 1.0 (14/14 alleles) in 1591C homozygotes and 0.130 (7/54 alleles) in normals (p < 0.0001). Haplotypes were discernible in all of the 1591C homozygotes and in several of the heterozygotes and normals. The CD4 162, TPI 2262A haplotype was found on only two of thirty-eight normal chromosomes, but was universally associated with 1591C. The data indicate that all TPI 1591C subjects are descendants of a common ancestor who probably lived in what is now England or France. The original mutation probably occurred well in excess of 1000 years ago.

Inferior vena cava thrombosis as a complication of femoral vein catheterisation.

Indwelling femoral venous catheters were prospectively studied by ultrasonography to define the frequency and evolution of inferior vena cava (IVC) thrombosis. IVC thrombosis was identified in six of 56 catheters (54 children). Only one patient with a positive ultrasound scan had clinical signs of thrombosis. All children with IVC thrombosis had had catheters in place for over six days. It is recommended that either the femoral central venous catheters are routinely changed at six days or ultrasound studies are routinely performed twice a week in all patients with catheters in situ for six or more days and that the catheter is removed immediately if evidence of thrombosis appears.

Thalassaemia and other haemoglobinopathies in general practice.

Haemoglobinopathies are now common in Australia following the migration of people from areas of high prevalence. This article gives practical advice on the management of patients incidentally found to be heterozygotes for the more common forms and also outlines complications of major haemoglobinopathies that are seen in family practice.

Homozygous beta-thalassaemia: a review of patients who had splenectomy at the Royal Alexandra Hospital for Children, Sydney.

Twenty-four patients with homozygous beta-thalassaemia who had been splenectomised and currently on treatment were studied retrospectively. They were divided into two groups. Group A: who had splenectomy prior to commencement of any regular blood transfusion. The mean haemoglobin for this group rose from 5.5 gm/dl pre-splenectomy to 7.7 gm/dl post splenectomy (p < 0.001). Group B: who were on regular blood transfusion when they had their splenectomy and the mean blood transfusion requirement dropped from 317 ml/kg/yr to 230 ml/kg/yr of packed red cells following splenectomy (p < 0.001). Three patients who were on regular blood transfusion and desferrioxamine developed Yersinia enterocolitica infection. They presented with fever and signs of an acute abdomen. At laparotomy, 2 of the patients had acute appendicitis. All 3 appendices grew Yersinia enterocolitica and one patient also had a Yersinia enterocolitica septicaemia. If a patient develops fever and enteritis, desferrioxamine should be stopped temporarily and cotrimoxazole started as prophylaxis against systemic Yersiniosis. No cases of pneumoccocal sepsis was reported.

Human triosephosphate isomerase deficiency resulting from mutation of Phe-240.

Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jó., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe]-->CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by disrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10-20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu]-->GAC [Asp]), which also results in thermolabile protein.

Colocalization of peptide and glucocorticoid receptor immunoreactivities in rat central amygdaloid nucleus.

The central amygdaloid nucleus (ACe) is part of the amygdaloid complex that participates in adrenocorticotrophin secretion, stress-related reactions and behavioral functions. The ACe contains numerous glucocorticoid receptor (GR)-immunoreactive (IR) neurons, and in addition it has been shown to contain several neuropeptide-IR somata and nerve terminals. In order to study the relationship between the GR- and neuropeptide-IR structures we mapped the distribution of GR-like immunoreactivity (LI) in amygdaloid complex and colocalized the neuropeptide- and GR-LIs in the ACe. In the amygdaloid complex the central, medial and cortical nuclei contained a high number of GR-IR neurons, whereas a moderate number of GR-IR neurons were observed in the basolateral and basomedial nuclei. Only a few GR-IR neurons were seen in the lateral nucleus. In the ACe, the majority of corticotrophin-releasing factor (CRF)-, met-enkephalin (met-ENK)-, neurotensin (NT)- and somatostatin (SOM)-IR neurons contained also GR-IR. About half of the substance P (SP)-IR neurons were seen to contain GR-IR, whereas only some of the few vasoactive intestinal polypeptide and cholecystokinin-IR neurons showed GR-LI. Nerve terminals containing calcitonin gene-related peptide and the above mentioned peptides were seen in close contact with the GR-IR neurons. These results suggest that the glucocorticoids may modulate directly the neurotransmitter synthesis of the CRF-, met-ENK, NT-, SOM- and SP-IR cells in the ACe.

Adenoidectomy in a girl with haemophilia.

Haemophilia A is a sex-linked inherited disease in which those affected are usually males, and females are usually asymptomatic carriers. This paper presents a haemophilia A carrier who has a low factor VIII level first, to remind readers that females can have low factor VIII levels with consequent increased tendency to bleed; second, to stress the importance of routine questioning regarding a bleeding tendency in the patient or the patient's family and third, to illustrate the importance of pre-operative diagnosis to allow adequate correction of the defect so that surgery such as adenoidectomy can be undertaken with safety in such a patient.

Molecular mechanisms associated with increased fetal hemoglobin G gamma-type in part-aboriginal family with beta thalassemia.

A part-Aboriginal family with beta thalassemia and raised hemoglobin F (HbF) was studied at the molecular level to determine if there were identifiable gene changes associated with increased production of HbF. Two beta thalassemia heterozygotes aged eight years and 18 months had raised HbF levels of 2.9% and 22% respectively. HbF was predominantly G gamma in composition. Five family members were typed for restriction fragment length polymorphisms (RFLPs) using nine restriction enzymes and five DNA probes specific for the beta globin cluster on chromosome 11. RFLPs were combined to construct haplotypes for the beta thalassemia and the high HbF defects. A beta globin subhaplotype comprising only 5' RFLP markers (-(+)-(+) +) co-segregated with the high HbF determinant. This has previously been associated with increased G gamma expression in beta thalassemia and sickle cell anemia. An additional Xmnl RFLP 5' to the G gamma gene, which has been described in individuals with elevated G gamma expression, was also demonstrated in those family members with increased G gamma levels. In this study both the 5' beta globin subhaplotype (-(+)-(+) +) and the Xmnl/gamma RFLP are present in the one family but the relative contributions of each cannot be determined.

Hemoglobin F production in heterocellular hereditary persistence of fetal hemoglobin and its linkage to the beta globin gene complex.

Some types of nondeletional heterocellular hereditary persistence of fetal hemoglobin (HPFH) appear to be caused by mutations in the beta globin gene cluster near the gamma globin genes, while in other cases the condition is associated with a gene or genes outside the beta globin gene complex. We have used DNA probes for chromosome 11 markers to localize the HPFH determinant in a large Australian kindred with nondeletional heterocellular HPFH. In 13 of the 58 family members studied the Hb F levels are increased to between 1.8% and 7.9%, the Hb F being composed predominantly of A gamma chains. All family members were typed for restriction fragment length polymorphisms detected by probes from the beta globin gene complex, and the nearby genetic markers D11S12, INS, and HRAS. Linkage analysis showed HPFH is closely linked to the beta globin gene cluster (confidence limits of theta, 0.0-0.19), D11S12 (theta, 0.0-0.23) and the insulin gene (theta, 0.0-0.11). These data and the gamma chain composition are consistent with HPFH in this family being caused by a mutation within the beta globin gene cluster.

Molecular defects in 2 examples of severe Hb H disease.

Severe Hb H disease presented in unexpected ways in 2 families of Greek origin. In 1, Hb H disease led to neonatal death. The underlying molecular defect was double-heterozygosity for the --Med/ alpha thalassaemia haplotype and a nondeletional alpha thalassaemia defect (alpha alpha T'Karditsa'/). The 2nd family requested antenatal diagnosis. The husband had mild nondeletional alpha thalassaemia. Initial investigations in the wife demonstrated unexpected gene mapping patterns. These have recently been shown to result from the (-alpha)Med 20.5/ haplotype.

A factor IX gene probe: its use in carrier detection, antenatal diagnosis and characterisation of the molecular basis for hemophilia B.

Of hemophilia B carriers, 67% were shown to have informative restriction fragment length polymorphisms (Taq I or Xmn I) associated with the factor IX gene. Analysis of DNA for these polymorphisms can enable the detection of the hemophilia B carrier state in females and of hemophilia B in the male fetus in the first trimester. Extensive mapping of the factor IX gene in one hemophiliac who developed antibodies to factor IX failed to detect structural abnormalities in his gene. A non-deletional basis for hemophilia B is proposed in this instance.