The national incidence and clinical picture of SLE in children in Australia - a report from the Australian Paediatric Surveillance Unit.

OBJECTIVES: The objectives of this paper are to prospectively determine the incidence of paediatric systemic lupus erythematosus (pSLE) in Australia as well as describe the demographics, clinical presentation and one-year outcome. STUDY DESIGN: Newly diagnosed cases of pSLE were ascertained prospectively from October 2009 to October 2011 through the Australian Paediatric Surveillance Unit (a national monthly surveillance scheme for notification of childhood rare diseases) as well as national subspecialty groups. Questionnaires were sent to notifying physicians at presentation and at one year. RESULTS: The annual incidence rate was 0.32 per 10(5) children aged less than 16 years. The incidence was significantly higher in children of Asian or Australian Aboriginal and Torres Strait Islander parents. Approximately one-third of children underwent a renal biopsy at presentation and 7% required dialysis initially although only one child had end-stage kidney disease (ESKD) at one-year follow-up. CONCLUSION: The incidence of pSLE in Australia is comparable to that worldwide with a significantly higher incidence seen in children of Asian and Australian Aboriginal and Torres Strait Islander backgrounds. Renal involvement is common but progression to ESKD, at least in the short term, is rare.

Frequency and significance of immediate contact reactions to peanut in peanut-sensitive children.

BACKGROUND: Parents of atopic children frequently report, and are alarmed by, contact reactions to foods. Some schools restrict foods due to concerns regarding possible systemic reactions following contact in allergic children. OBJECTIVE: We aimed to determine the frequency with which peanut-sensitive children exhibited contact sensitivity to peanut butter and to assess the significance of such reactions. METHODS: One gram of peanut butter was applied directly to the skin of 281 children who were skin prick test (SPT) positive to peanut (immediate skin application food test; I-SAFT). The test was considered positive if one or more weals were present when the patch was removed after 15 min. A subset of children then underwent an open-label oral challenge with graded amounts of peanut protein. RESULTS: During 3515 clinic visits, 330 I-SAFT tests for peanut contact sensitivity were performed; 136 (41%) were positive. The mean SPT diameter was 10 mm in the I-SAFT-positive children and 8.5 mm in the I-SAFT-negative children (t-test, P<0.0001). No child had a systemic reaction following topical application of peanut butter. Eighty-four children had 85 oral challenges after blinded, placebo-controlled I-SAFT testing. Challenge was positive in 26/32 of those with a positive I-SAFT and negative in only 6/32. Challenge was also positive in 26/53 but negative in 27/53 of those with a negative I-SAFT (sensitivity 50%, specificity 82%, chi2, P=0.003). CONCLUSION: A minority of children sensitized to peanut (positive SPT) develop localized urticaria from prolonged skin contact with peanut butter. No tested subjects, including ones with systemic reactions upon oral challenge, developed a systemic reaction to prolonged skin exposure to peanut. Therefore, systemic reactions resulting from this mode of contact with peanut butter appear highly unlikely.

Adherence issues in children and adolescents receiving highly active antiretroviral therapy.

The introduction of highly active antiretroviral therapies (HAART) for the treatment of paediatric HIV infection poses additional adherence challenges for children and families living with HIV A preliminary survey of 18 parents of children receiving HAART in Australia showed that although parents report high level of child adherence to HAART, specific features of the medication regimen, such as taste and number of medications made administration of HAART extremely difficult.Moreover, interaction between the treatment regimen and the day-to-day lives of families increases the adherence challenge. While some agreement exists in relation to the concerns families have about negative aspects of HAART; the diversity of issues suggests the need for ongoing and individualized support and information to families.

Use of interventions for reducing mother-to-child transmission of HIV in Australia.

OBJECTIVE: To describe the extent and outcome of use of interventions for reducing the risk of HIV transmission from mother to child in Australia. DESIGN: National surveillance for perinatal exposure to HIV. PARTICIPANTS AND SETTING: Notified cases of HIV infection in women in Australia and their perinatally exposed children, 1982-1999. OUTCOME MEASURES: Trends over time in use of interventions (antiretroviral therapy in pregnancy, elective caesarean delivery and avoidance of breastfeeding) and perinatally acquired HIV infection. RESULTS: By 31 March 2000, 204 children were reported as having been born in 1982-1999 to 162 women whose HIV infection had been diagnosed by 31 December 1999. The child's HIV infection status was established for 182 (89.2%); the mother's HIV infection was diagnosed antenatally in 91 of these cases (50%). Among women diagnosed antenatally, use of elective caesarean delivery and antiretroviral therapy in pregnancy increased significantly, from 3% and 14% by women whose children were born in 1982-1993, to 21% (P=0.01) and 88% (P<0.001), respectively, by women whose children were born in 1994-1999. Most women (95%) diagnosed antenatally avoided breastfeeding their children. The percentage of infected children born to women diagnosed antenatally declined from 26% among children born in 1982-1993 to 19% among those born in 1994-1999. The percentage of infected children was significantly lower among those whose mothers used antiretroviral therapy in pregnancy (11% versus 36%; P=0.03). CONCLUSION: Antiretroviral use in pregnancy, elective caesarean delivery and avoidance of breastfeeding have been effective interventions for reducing the risk of mother-to-child HIV transmission in Australia. While the rate of perinatal HIV transmission has declined, it remains high in comparison with rates reported from other industrialised countries.

Reduced memory B-cell populations in boys with B-cell dysfunction after bone marrow transplantation for X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is a lethal disease resulting in death in infancy. In many instances, haploidentical bone marrow transplantation (BMT) offers reconstitution of T-cell immunity alone, with residual hypogammaglobulinaemia. The exact nature of B-cell dysfunction in these patients is unclear, although differentiation arrest of the B cells is a potential explanation. To ascertain the differentiation status of peripheral blood B lymphocytes from XSCID patients after BMT, the surface expression of CD19, CD10, CD34, CD5, serum immunoglogulin (sIg)M, sIgD, sIgG and CD27 on these B cells was investigated using three-colour flow cytometry. CD27 is a marker of memory B cells. Populations of CD19+IgM-D- B cells, CD19+IgM-only, CD19+IgG+CD27+ and CD19+IgM+ CD27+ B cells were found to be diminished in the XSCID patients after BMT with persistent hypogammaglobulinaemia, compared with both post-BMT patients with B-cell function and age-matched normal controls. This indicated the lack of CD19+IgM-D- B cells, which represent Ig isotype-switched B cells, as well as CD19+IgM-only and CD19+IgG+CD27+ or CD19+IgM+CD27+ memory B-cell populations. Interaction between CD27 and its ligand CD70 has been shown to induce IgG and IgM production by CD27+ B cells. Therefore, the lack of CD27/70 interaction is a probable explanation for the hypogammaglobulinaemia in these patients after BMT.

Caspase-3 activates endo-exonuclease: further evidence for a role of the nuclease in apoptosis.

Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130-185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 microM etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells.

Caspase-3-dependent and caspase-3-independent pathways leading to chromatin DNA fragmentation in HL-60 cells.

Apoptosis induced by etoposide (VP-16) in HL-60 cells was confirmed to be caspase-dependent. It was fully inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk. However, the caspase-3-specific inhibitor Z-DEVDfmk only partially inhibited apoptosis. This indicated that a second caspase is required in vivo for full activation of the apoptotic nucease CAD. Aurin tricarboxylic acid (ATA) did not inhibit VP-16-induced apoptosis. In contrast, apoptosis induced by hydroxychloroquine (HCQ) in HL-60 cells was caspase-3 independent and was fully inhibited by ATA. Thus, CAD does not appear to be involved in chromatin DNA degradation in this case. A second apoptotic nuclease is postulated to degrade the DNA, likely endo- exonuclease, an abundant nuclear enzyme that acts on both DNA and RNA and is present in latent form. HCQ, but not VP-16, stimulated DNA degradation ("laddering") in isolated nuclei. This indicates that the drug can act directly in the nuclei to trigger activation of the second latent apoptotic nuclease.

Identification of X-linked severe combined immunodeficiency by mutation analysis of blood and hair roots.

Severe combined immunodeficiency is a heterogenous syndrome of varied genetic origins of which the X-linked type is the commonest (XSCID). The most sensitive method for diagnosis of XSCID in the absence of X-linked inheritance pattern is by mutation analysis. In this report we have performed mutation analysis in 13 unrelated boys transplanted (BMT) for SCID without a known cause to determine the frequency of XSCID. Five boys had an affected male relative. We also assessed the utility of hair roots for children without pre-transplant blood stored for mutation analysis since donor genotype was expressed in peripheral blood post BMT. Screening was performed by analysis of single-strand conformational polymorphism (SSCP) followed by sequencing of candidate exons. Mutations were found in 11 cases, of which six were sporadic, and maternal mosaicism was found in one family. Three mothers of the six sporadic cases were identified as carriers. The majority (6/8) of boys with SCID had gammac deficiency despite the absence of X-linked inheritance pattern. The significant frequency of de novo mutations and the occurrence of maternal germline mosaicism highlights the importance of mutation analysis. The strategy of using DNA from hair roots was particularly valuable where no pre-transplant blood was stored. Characterization of the mutations will also enable research into the correction of these genetic defects.

Unravelling age effects and sex differences in needle pain: ratings of sensory intensity and unpleasantness of venipuncture pain by children and their parents.

Age and sex differences were investigated in children's self-report of venipuncture pain. Equal numbers of boys and girls aged 3-15 years (n = 110) made separate ratings of the intensity and unpleasantness of their needle pain, using a paired Visual Analogue Scale (VAS) technique. The parents of these children used the same method to give ratings of predicted pain and unpleasantness before the needle, as well as ratings based on observing their child during the needle. Results showed that, across age, children's intensity and unpleasantness scores were highly correlated (r = 0.78), and that both of these ratings decreased with increasing age. Analyses of covariance showed that, with the variance in the unpleasantness ratings accounted for, a significant age main effect persisted for the intensity ratings (scores decreasing with increasing age), with no effect of sex. In the corollary analysis, with intensity scores entered as a covariate, unpleasantness ratings showed no main effect of age, but a significant main effect of sex emerged: girls' ratings of pain unpleasantness, when averaged across age, were significantly higher than boys'. The interaction between age and sex was explored in analysis of the relative difference between intensity and unpleasantness ratings. The results indicated that, from approximately 8-years of age, children (especially girls) gave significantly higher ratings of unpleasantness than sensory intensity of needle pain. Prior to the age of 8 years, children tended to give equivalent ratings of intensity and unpleasantness, with no evidence of a sex difference. The agreement between parental and children's ratings was higher for parents' observed, as opposed to predicted, scores, especially for pain intensity, with no systematic influence of the child's age and sex. In conclusion, it is suggested that age effects in children's self-report of needle pain are predominantly manifest in ratings of sensory intensity, whilst sex effects are predominantly manifest in ratings of an affective (unpleasantness) dimension. It is argued that both age and sex differences are largely the function of pain reporting variables, rather than reflecting fundamental age or sex based variance in nociceptive processing.

Current concepts of the actions of paracetamol (acetaminophen) and NSAIDs.

There is much uncertainty about the mechanism of action of paracetamol (acetaminophen). It is commonly stated that, unlike the non-steroidal anti-inflammatory drugs (NSAIDs), it is a weak inhibitor of the synthesis of prostaglandins. This conclusion is made largely from studies in which the synthesis of prostaglandins was measured in homogenized tissues. However, in several cellular systems, paracetamol is an inhibitor of the synthesis of prostaglandins with IC(50) values ranging from approximately 4 microM to 200 microM. Paracetamol is not bound significantly to plasma proteins and therefore the concentrations in plasma can be equated directly with those used in in vitro experiments. After oral doses of 1 g, the peak plasma concentrations of paracetamol are approximately 100 microM and the plasma concentrations are therefore in the range where marked inhibition of the synthesis of prostaglandins should occur in some cells. Paracetamol is metabolized by the peroxidase component of prostaglandin H synthase but the relationship of this to inhibition of the cyclooxygenase or peroxidase activities of the enzyme is unclear. Paracetamol is also metabolized by several other peroxidases, including myeloperoxidase, the enzyme in neutrophils which is responsible for the production of hypochlorous acid (HOCl). The metabolism of paracetamol by myeloperoxidase leads to the decreased total production of HOC1 by both intact neutrophils and isolated myeloperoxidase, even though the initial rate of production of HOC1 is increased. The IC(50) value, derived from inhibition of the total production of HOC1 by isolated myeloperoxidase, is 81 microM. Several NSAIDs inhibit functions of neutrophils in media containing low concentrations of protein but their effects, in contrast to that of paracetamol, are generally produced only at concentrations greater than those of the unbound drug in plasma during treatment with the NSAIDs. However, neutrophils isolated during treatment with NSAIDs, such as piroxicam, ibuprofen and indomethacin show decreased function. Paracetamol has little or no anti-inflammatory activity by itself but may potentiate the clinical activity of NSAIDs in the treatment of rheumatoid arthritis.

Carrier identification in X-linked immunodeficiency diseases.

OBJECTIVE: Carrier identification in X-linked immunodeficiency disorders can be based on the demonstration of non-random X inactivation (NRXI) in affected blood cell lineages when growth is impaired in cells expressing the abnormal gene. We examined the utility of seeking evidence of NRXI to test the carrier status of women in families affected by X-linked severe combined immunodeficiency (XSCID) and X-linked hypogammaglobulinaemia (XLH), to identify as carriers the mothers of boys with SCID or hypogammaglobulinaemia whose phenotype suggested X-linkage and to infer X-linkage in boys with SCID or hypogammaglobulinaemia whose disease was not clearly X-linked on the basis either of family history or clinical and immunological characteristics. METHODOLOGY: A polymerase chain reaction-based method was used to amplify a polymorphic CAG repeat in the first exon of the androgen receptor gene after selective digestion of the active X chromosome with a methylation-sensitive enzyme, HpaII to distinguish between the paternal and maternal alleles and to identify their methylation status. RESULTS: Heterozygosity was found in 24 of 31 female subjects (77%). As anticipated, NRXI could be demonstrated in all lymphoid cells studied from obligate carriers of XSCID and an obligate carrier of XLH but not on a carrier of X-linked immunodeficiency with hyper-IgM. The finding of NRXI in the mother of a boy with a SCID variant showed her to be a carrier of XSCID and establishes that her son has XSCID, not otherwise evident from available data. CONCLUSIONS: This PCR assay provides a rapid method for carrier detection of X-linked immunodeficiencies, and has allowed us to expand the phenotype of XSCID

Acquired autoimmune thrombocytopenia post-bone marrow transplantation for severe combined immunodeficiency.

Children with severe combined immunodeficiency (SCID) have profoundly diminished humoral and cellular immunity resulting in death during infancy unless immune reconstitution occurs by bone marrow transplantation (BMT). Thrombocytopenia post-bone marrow transplantation can be seen in relation to infection, graft-versus-host disease (GVHD) and rarely, as an autoimmune phenomenon due to immune dysregulation. We report two cases of severe AITP following BMT for SCID. Both cases developed large intracerebral hemorrhages from which one died. Autoimmune thrombocytopenia in this setting can be life-threatening and we recommend early and active intervention.

Pharmacology of benzydamine.

Benzydamine is a topical anti-inflammatory drug which is widely available and used topically for the treatment of the mouth. It is also used as a gel for application to inflamed joints. It has physicochemical properties and pharmacological activities which differ markedly from those of the aspirin-line non-steroidal anti-inflammatory drugs. Benzydamine is a weak base unlike the aspirin-like drugs which are acids or metabolized to acids. A major contrast with the aspirin-like drugs is that benzydamine is a weak inhibitor of the synthesis of prostaglandins but it has several properties which may contribute to its anti-inflammatory activity. These properties include inhibition of the synthesis of the inflammatory cytokine, tumour necrosis factor-alpha (EC(50), 25 micromol/L). Inhibition of the oxidative burst of neutrophils occurs under some conditions at concentrations of 30 to 100 micromol/L, concentrations which may be produced within oral tissues after local application. A further activity of benzydamine is a general activity known as membrane stabilization which is demonstrated by several actions including inhibition of granule release from neutrophils at concentrations ranging from 3 to 30 micromol/L and stabilization of lysosomes. Lack of knowledge of the tissue concentrations of benzydamine limit the correlation between pharmacological activities in vitro and in vivo. The concentration of benzydamine in the mouthwash is 4 mmol/L but the concentrations in oral tissues have not been studied adequately. Limited data in the rat indicates that concentrations of benzydamine in oral tissues are approximately 100 micromol/L.

An investigation of a possible role for mitochondrial nuclease in apoptosis.

Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 microM) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0-1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation.

Paediatric HIV update.

Less than half of the paediatric HIV infections recorded in Australia have resulted from perinatal transmission, but in recent years this has been the predominant mode of infection. There are 136 infants who are known to have been exposed perinatally to HIV in Australia: 49 of these are infected. Caesarean section is thought now not to reduce the risk of perinatal transmission (PNT); rather, the risk increases with duration of membrane rupture and rises rapidly after 4 h of membrane rupture. However, no data exist to show that interventions to expediate delivery after membrane rupture reduce the risk of PNT. Data such as these suggest that the majority of perinatal infections (probably about 60%) occur close to the time of delivery. While the overall risk of PNT for non-breast fed infants is approximately 20-25%, the risk of infection for the infant is considerably increased when there is evidence of increased maternal viral burden. Advanced maternal disease predicts that if the infant is infected there is more likely to be early progression of HIV than is the case for the less frequently infected infants of mothers who are asymptomatic. Bottle feeding may prevent infection of 10% of children exposed perinatally. Use of zidovudine by the mother in the third trimester and i.v. zidovudine during labour, followed by oral zidovudine for the infant for 6 weeks can reduce the PNT rate by two thirds, to about 8%. Approximately 3% of uninfected infants with perinatal HIV exposure may be found to be transiently virus positive but eventually become antibody negative and thus appear to have eliminated the virus. The risk of Pneumocystis carinii pneumonitis (PCP) cannot be predicted on the basis of CD4 count and it is recommended that all children of infected mothers commence PCP prophylaxis around the age of 6 weeks-2 months and continue that therapy until the age of 12 months or until it becomes clear that the infant is uninfected. The cumulative risk of AIDS increases rapidly during the first year of life to about 20%, then more slowly at a rate of about 2 or 3% a year. The shape of this curve reveals the bimodal progression of HIV disease in children. About 15-20% of children rapidly develop a severe immune deficiency, opportunistic infections and, in most cases, encephalopathy. There is a very high morbidity rate in this group of children, most of whom die before the age of 3 or 4 years. In contrast, 80-85% of children only become immunodeficient after a relatively long period, which is similar to or perhaps even longer than that in adults. Recent studies indicate that zidovudine antiviral monotherapy is no longer appropriate. While no clear alternative to monotherapy has emerged most would, wherever possible, commence antiretroviral therapy with a combination of two or three drugs including zidovudine plus didanosine or lamivudine. If a third drug is used it would probably be a protease inhibitor.

An investigation of the placebo effect and age-related factors in the report of needle pain from venipuncture in children.

To examine the potential role for a placebo cream in reducing reported needle pain severity in children, and the impact of age-related factors on pain self-report, a convenience sample of 117 children scheduled for venipuncture were randomly assigned to one of three treatments: (a) placebo cream with the suggestion that it might help reduce needle pain, (b) placebo cream with no indication as to the cream's purpose, and (c) no cream (control group). In allocation to treatment, children were stratified by age group, (3-7, 8-11, 12-17 years). They rated their needle pain severity (both predicted and reported) using the Faces Pain Scale, and rated their anxiety about the procedure using the Children's Anxiety and Pain Scale. Children in the cream groups were also asked whether they thought the cream had helped. Using video-tapes, an independent observer, blind to the placebo manipulation, rated each child's reaction to the needle. For the two groups receiving cream, 83% of those children told it might help stated that they believed it did, as compared with only 33% of children who received the cream but were told nothing of its purpose. These beliefs, however, were not reflected in self-report ratings of pain which showed no statistically significant treatment effect. Similarly, children who gave higher preprocedural anxiety ratings were no more likely to report less pain as a result of receiving the cream. There was, however, a treatment effect on the observer's ratings: children receiving cream plus suggestion were assigned significantly lower ratings of pain-related behaviour than those children who received the cream alone. While venipuncture was associated with only mild levels of pain, younger children, irrespective of treatment group, did report more pain than older children. Hierarchical regression analysis indicated that 60% of the variance in self-reported pain severity scores could be accounted for by how much the child thought the needle would hurt, how anxious the child was about receiving the needle, gender (higher pain ratings associated with girls), and estimated body surface area (higher pain ratings associated with smaller bodies). We conclude that the efficacy of placebo treatments for needle pain in children may depend on the suggestion of a possible benefit rather than upon treatment application per se.

Induction of apoptosis in peripheral blood lymphocytes following treatment in vitro with hydroxychloroquine.

OBJECTIVE: Defective regulation of apoptosis may be central to the development of autoimmune disorders. This study investigated the possibility that the antirheumatic effect of hydroxycholoroquine (HCQ) may be achieved by up-regulation of apoptosis. METHODS: Peripheral blood lymphocytes collected from normal controls and patients with systemic lupus erythematosus (SLE) were cultured in the presence or absence of a range of concentrations of HCQ. Cells undergoing apoptosis were identified by several standard methods, including morphologic changes, DNA fragmentation, and flow cytometry. For some experiments, lymphocytes were simultaneously stained with antibodies to T cell surface markers and with propidium iodide for dual-stain flow cytometric studies. RESULTS: HCQ was able to induce apoptosis in peripheral blood lymphocytes in a dose- and time-dependent manner. HCQ induced these changes in all T cell subpopulations studied. There was no significant difference between the controls and patients with SLE in terms of the percentage of apoptotic cells detected following treatment with HCQ. CONCLUSION: The present study demonstrated that HCQ induces apoptosis in peripheral blood lymphocytes, which leads to the speculation that HCQ may exert its antirheumatic effect through this mechanism.