Pancreatitis and adenoviral infection in children after blood and marrow transplantation.

Pancreatitis is a well-recognized consequence of blood and marrow transplantation (BMT). In a 4-year period, between January 2001 and December 2004, five children who received a BMT in our institution were diagnosed as having pancreatitis. Four of these five children also had adenoviral infection. We report these four cases and highlight the importance of investigating for pancreatitis patients who have any abdominal symptoms post BMT, and include specific stool culture for viral isolation, if it is not already known.

Cytomegalovirus antiviral resistance associated with treatment induced UL97 (protein kinase) and UL54 (DNA polymerase) mutations.

HCMV-related illness due to infections with antiviral resistant virus was verified by phenotypic and genotypic assays in 17% (8/47) of high-risk immunocompromised Australian patients. Selective PCR-sequencing of UL97 (protein kinase; PK) and UL54 (DNA polymerase; DNApol) regions important for antiviral sensitivity, identified the majority (6/8) of resistant strains through detection of mutations known to confer antiviral resistance. Additionally, eight UL54 (DNApol) mutations (N408K, T691S, A692V, S695T, L737M, A834P, V955I, and A972V) of unknown phenotype were identified in six specimens from patients with clinical evidence of antiviral resistant infections. One isolate was resistant to ganciclovir (GCV) and another resistant to PFA on phenotypic testing where mutations in UL97 (PK) or UL54 (DNApol) were not detected, suggesting a loss of correlation between phenotype and genotype. Selective PCR-sequencing of UL97 (PK) and UL54 (DNApol) provided rapid and comprehensive results, but missed some resistance detected by phenotypic assays. A combination of phenotypic and genotypic assays is recommended for complete analysis of CMV antiviral resistance, as well as further definition of the clinical relationship between novel UL54 (DNApol) mutations and antiviral resistance.

Diagnosis and management of paediatric hepatitis C virus infection.

Hepatitis C virus (HCV) infection in children is uncommon and there are few guidelines indicating optimal management. It is estimated that 125-250 children are infected vertically with HCV in Australia each year and very few of these children are diagnosed and followed medically. Without accurate diagnosis and follow up, these children cannot be offered optimal care, and are at risk of presenting in adult life with significant liver pathology and long-term sequelae.

Transcriptional regulation of major histocompatibility complex class I by flavivirus West Nile is dependent on NF-kappaB activation.

Infection by the flavivirus West Nile (WNV) is associated with a virus-specific increase of major histocompatibility complex class I (MHC-I) molecules on the cell surface of diploid vertebrate cells. The increased MHC-I cell surface expression is functional and is associated with increased susceptibility to secondary WNV-immune and alloimmune cytotoxic T cells. WNV-induced up-regulation of cell surface MHC-I expression is associated with NF-kappaB activation and increased transcription of MHC-I mRNA. WNV infection increases luciferase activity of RAWa4 long terminal repeat (LTR) cells, which are transfected stably with a plasmid containing 2 NF-kappaB binding sites, the human immunodeficiency virus LTR linked to a luciferase reporter gene. The NF-kappaB-induced complexes are a p50/p65 heterodimer and another faster migrating species containing p50 homodimers. WNV-induced activation of NF-kappaB and the up-regulation of MHC-I were blocked by the protein kinase C inhibitor H-7 and salicylate, both of which block phosphorylation of inhibitor kappaB.

Management of neonatal herpes simplex virus infection.

Herpes simplex viruses (HSV) are ubiquitous pathogens which can be transmitted vertically causing significant morbidity and mortality in neonates. Neonatal HSV infection is infrequent with an incidence ranging from 1 in 3,500 to 1 in 20,000, depending on the population. Neonatal HSV infection is much more frequent in infants born to mothers experiencing a primary HSV infection with an incidence approaching 50%, while infants born to mothers experiencing recurrent HSV infection have an incidence of less than 3%. Neonatal infections are clinically categorised according to the extent of the disease. They are: (i) skin, eye and mouth (SEM) infections; (ii) central nervous system infection (encephalitis)--neonatal encephalitis can include SEM infections; and (iii) disseminated infection involving several organs, including the liver, lung, skin and/or adrenals. The central nervous system may also be involved in disseminated infections. Caesarean section, where the amniotic membranes are intact or have been ruptured for less than 4 hours, is recommended for those women who have clinical evidence of active herpes lesions on the cervix or vulva at the time of labour. This procedure significantly decreases the risk of transmission to the infant. Diagnosis of neonatal infection requires a very high level of clinical awareness as only a minority of mothers will have a history of genital HSV infection even though they are infected. Careful physical examination and appropriate investigations of the infant should accurately identify the infection in the majority of cases. Treatment is recommended where diagnosis is confirmed or there is a high level of suspicion. The current recommendation for treatment is aciclovir 20 mg/kg 3 times daily by intravenous infusion. Careful monitoring of hydration and renal function as well as meticulous supportive care of a very sick infant is also required. The newer anti-herpes agents, valaciclovir and famciclovir, offer no advantage over aciclovir and are not recommended for neonatal HSV infection. Prognosis is dependent upon the extent of disease and the efficacy of treatment, with highest rates of morbidity and mortality in disseminated infections, followed by central nervous system infection and the least in SEM infection. However, SEM infection is associated with poor developmental outcome even in infants who do not have encephalitis. Studies to improve the outcome of SEM infection are in progress. Neonatal HSV infections, although being relatively uncommon, are associated with significant morbidity and mortality if unrecognised and specific treatment is delayed. Diagnosis relies on a high level of clinical suspicion and appropriate investigation. With early therapy, the prognosis for this infection is considerably improved.

The use of flow cytometry to detect antiviral resistance in human cytomegalovirus.

A method for detecting the antiviral susceptibility of human cytomegalovirus (HCMV) isolates to antiviral agents using flow cytometry was developed. This method has been used to detect the resistance phenotype of HCMV isolates to ganciclovir (GCV). The procedure involves infecting MRC-5 cells with 10(4) pfu HCMV for 120 h, then fixing and permeabilising the cells to allow intracellular labelling of the HCMV early and late antigens. The percentage reduction in the fluorescence positive population of HCMV-infected MRC-5 cells treated with GCV at concentrations of 20 or 50 microM compared with control cultures without GCV was determined. The IC50 defined as a < 50% reduction in the fluorescence positive population in cells infected in the presence of 20 microM GCV or an IC90 defined as a < 90% reduction in the fluorescence-positive population in cells infected in the presence of 50 microM GCV, correlated with resistance determined by a plaque reduction assay. The FACS assay is a rapid and reproducible method for detecting antiviral resistance of HCMV.

Use of aciclovir in herpes simplex virus infections.

Herpes simplex virus type 1 and type 2 cause a wide range of illnesses ranging from minor cold sores to severe necrotising encephalitis or disseminated systemic infections seen in immunocompromised patients including neonates. Following primary infection, the virus is not eradicated from the body but is latent in sensory nerve ganglia where it can reactivate and cause recurrent disease. Aciclovir is the most studied and used antiviral agent with activity against herpes simplex virus infections. In most situations the use of aciclovir shortens the duration of clinical illness and viral shedding and reduces morbidity and mortality. All life- or sight-threatening infections should be managed in an inpatient hospital setting with intravenous therapy. The use of oral aciclovir is recommended in patients with non-life-threatening illness who may still have significant symptoms.

Differential tropism and chemokine receptor expression of human immunodeficiency virus type 1 in neonatal monocytes, monocyte-derived macrophages, and placental macrophages.

Laboratory-adapted (LA) macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1) isolates (e.g., HIV-1(Ba-L)) and low-passage primary (PR) isolates differed markedly in tropism for syngeneic neonatal monocytes, monocyte-derived macrophages (MDMs), and placental macrophages (PMs). Newly adherent neonatal monocytes and cultured PMs were highly refractory to infection with PR HIV-1 isolates yet were permissive for LA M-tropic isolates. Day 4 MDMs were also permissive for LA M-tropic isolates and additionally, were permissive for over half the PR isolates tested. Qualitative differences in PR HIV-1 infection of monocytes/MDMs could not be correlated with CD4 levels alone, and in all three cell types the block to PR HIV-1 strain replication preceded reverse transcription. Neonatal monocyte susceptibility to PR HIV-1 strains correlated with increasing CCR-5 expression during maturation. CCR-5 could not be detected on newly adherent (day 1) neonatal monocytes, in contrast to adult monocytes (H. Naif et al., J. Virol. 72:830-836, 1998), but was readily detectable after 4 to 7 days of culture. However, moderate CCR-5 mRNA levels were present in day 1 neonatal monocytes and remained constant during monocyte maturation. CCR-5 was not detectable on the surface of PMs, yet the receptor was present within permeabilized cells. Notably, two brain-derived PR HIV-1 isolates from a single patient, differing in their V3 loops, were discordant in their abilities to infect neonatal monocytes/MDMs and PMs, yet both isolates could infect newly adherent adult monocytes. Together these data strongly suggest that LA HIV-1 isolates are able to infect neonatal monocytes at earlier stages of maturation and lower-level expression of CCR-5 than PR isolates. The differences between neonatal and adult monocytes in susceptibility to PR isolates may also be related to the level of CCR-5 expression.

Progressive vaccinia treated with ribavirin and vaccinia immune globulin.

A 67-year-old man with metastatic melanoma and chronic lymphocytic leukemia was inadvertently given a vaccinia melanoma oncolysate vaccination. He developed progressive vaccinia at the site of inoculation. The lesion started to heal only when he was treated with ribavirin. Vaccinia immune globulin was administered and appeared to help control the initial lesion and limit the development of satellite lesions.

Advances in antiviral therapy.

For many years, acyclovir has been used to treat herpes simplex and varicella zoster infections in adults and children, although new drugs with improved bioavailability and dosage regimens (ie, famciclovir, valaciclovir) are replacing it for the outpatient management of these conditions in adults. Acyclovir remains the treatment of choice for severe herpes infections in both immunocompetent and immunosuppressed patients. Data on the newer antiherpes drugs in children are not available. Treatment of severe cytomegalovirus infections with ganciclovir and foscarnet is difficult because of toxicity; whether improved formulations of these drugs or newer agents prove clinically useful remains to be seen. For the most part, treatment of other herpesviruses is not indicated. The major advance in pediatric HIV treatment is the reduction in vertical transmission with peripartum zidovudine, although the optimal use of antiretrovirals in this situation remains to be determined. The nucleoside analogues zidovudine, zalcitabine, didanosine, and stavudine have been assessed in HIV-infected children; pediatric data about appropriate combinations (eg, with the protease inhibitors and the nonnucleoside reverse transcriptase inhibitors) and dosage regimens lag well behind the adult literature. The effectiveness of ribavirin in respiratory syncytial virus disease is uncertain. Preliminary data suggest that interferons may have a role in the management of chronic hepatitis B and C.

Acyclovir for the prevention and treatment of varicella zoster in children, adolescents and pregnancy.

Varicella causes a mild, self-limiting childhood disease that may reactivate years later as shingles. In immunocompromised patients with altered cell mediated immunity, and rarely in healthy individuals, varicella results in a life-threatening infection. The antiviral drug, acyclovir, substantially reduces the mortality and risk of severe disease in these groups of patients. Early commencement of acyclovir is recommended for children with both varicella and altered cell mediated immunity, newborns during the first 2 weeks of life, preterm infants in the neonatal nursery, and severe varicella or shingles (including ocular zoster) in any patient, as well as during pregnancy. Acyclovir may be considered in children with serious cardiopulmonary disease or chronic skin disorders where varicella may exacerbate the underlying disease or increase the risk of secondary bacterial sepsis. Acyclovir, however, is not recommended for healthy individuals without severe disease, as a prophylactic agent against varicella, for asthmatics receiving aerosolized or low-dose oral steroids and/or as treatment of the post-varicella syndromes. When acyclovir is prescribed it should be given intravenously to those with severe disease, those at risk of dissemination and in children younger than 2 years of age.

Herpes simplex virus protein targets for CD4 and CD8 lymphocyte cytotoxicity in cultured epidermal keratinocytes treated with interferon-gamma.

In early recurrent herpetic lesions, CD4 T lymphocytes are the predominant infiltrating cells, and keratinocytes expressing major histocompatibility complex (MHC) class II antigens, induced by interferon-gamma (IFN-gamma), are the major site of herpes simplex virus (HSV) replication. IFN-gamma pretreatment of human keratinocytes in vitro reduced MHC class I antigen down-regulation by HSV-1 infection and induced expression of HLA-DR that was unaltered by subsequent HSV-1 infection. Incubation of these infected keratinocytes with phosphonoacetic acid (PAA) almost completely inhibited expression of four major HSV glycoproteins, although expression of early proteins was not affected. Weak CD8 T lymphocyte cytotoxicity against IFN-gamma-stimulated, HLA-DR-expressing HSV-1-infected keratinocytes was consistently directed to the immediate early/early proteins (all 9 patients tested) but against late proteins to a lesser degree (4/9 patients). However, CD4 T lymphocyte cytotoxicity was much greater and directed predominantly against late HSV-1 glycoproteins (all 9 subjects tested) in these cells.

HIV infection of placental macrophages: their potential role in vertical transmission.

Placental macrophages were isolated and cultured in vitro to investigate their susceptibility to HIV infection and possible role in vertical transmission of HIV. After 10 days of in vitro culture the cells were positive for nonspecific esterase and acid phosphatase and negative for myeloperoxidase and placental alkaline phosphatase. They expressed cell surface HLA-ABC, HLA-DR, CD45, as well as CD68 intracellularly, as detected by flow cytometry, confirming their macrophage lineage. Approximately 80% of cells expressed surface CD14. CD4 antigen was expressed at very low levels and was confirmed by antibody blocking experiments. Infection of placental macrophage cultures with HIV resulted in a transient peak of viral replication 3 to 7 days after infection, but no later rise in HIV was detected with culture of up to 60 days. HIV replication was not up-regulated by coculture with phytohemagglutinin-stimulated lymphocytes or by treating infected cultures with tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor.

CTL recognition of west Nile virus-infected fibroblasts is cell cycle dependent and is associated with virus-induced increases in class I MHC antigen expression.

Confluent and non-confluent mouse embryo fibroblast (CMEF and NCMEF) monolayers were infected with West Nile virus (WNV) for 24 hr, and class I major histocompatibility complex antigen (MHC-I) concentrations measured by flow cytometry (FCM). Concentrations on CMEF increased significantly more than on NCMEF. This was not owing to differences in interferon-beta (IFN-beta)-mediated MHC induction, as the IFN-beta quantity secreted by each infected fibroblast was similar in each culture, and IFN-beta-mediated MHC-I induction on NCMEF was greater than on CMEF. Furthermore, despite neutralization of WNV-induced supernatant IFN-beta, CMEF increased MHC-I expression significantly more than NCMEF. Functionally, WNV-infected CMEF were lysed 10-fold better by WNV-specific and allospecific cytotoxic T cells, than infected NCMEF. FCM demonstrated 76% CMEF and 68% NCMEF distributed in G0/G1. This represented G0 in CMEF, and G1 in NCMEF, confirmed by ribonucleotide reductase M1 subunit labelling, where only 20% CMEF was labelled, compared to 84% NCMEF. The possible implications for antiviral immune responses are discussed.

Human immunodeficiency virus type 1 infection of human placental macrophages in vitro.

Placental macrophages (Hofbauer cells) were isolated and cultured in vitro to investigate their susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Of adherent cells, 80% expressed CD14, and > 99% were nonspecific esterase-positive. CD4 antigen was expressed at very low levels. CD4 mRNA could be detected in the cells by reverse transcription followed by polymerase chain reaction. The macrophages were infected productively after inoculation with low-passage blood isolates of cell-free HIV-1. Peak virus titers were detected 3-7 days after infection by HIV-1 antigen ELISA and reverse transcriptase assay. Replication of HIV-1 in placental macrophages was less than in blood monocytes. HIV-1 RNA was detected in placental macrophages by in situ hybridization 16 days after infection. Multinucleated giant cells were identified in some cultures, indicative of an HIV-induced cytopathic effect. Thus, placental macrophages can be infected productively with clinical isolates of HIV-1, and such cells may act as a reservoir of virus for transmission to the fetus in utero.

Uptake of extracellular Ca2+ is a requirement for production of interferon-gamma by cord blood mononuclear cells.

We studied the response of adult peripheral and cord blood mononuclear cells (MNCs) to graded concentrations of phytohemagglutinin (PHA) to characterize the requirements for proliferation and interferon-gamma (IFN-gamma) production. As the concentration of PHA was decreased, the proliferative response of both adult and cord blood MNCs decreased in parallel. At high concentrations of PHA (a: 120) cord blood MNCs display normal high proliferation but IFN-gamma production was greatly diminished compared with adult MNCs. Addition of the calcium ionophore, A23187, to PHA-stimulated adult MNCs had no effect on proliferation or IFN-gamma production at any of the concentrations of PHA tested. Furthermore, A23187 alone had no effect on proliferation or IFN-gamma secretion. Similarly, the addition of A23187 to PHA-stimulated cord-blood MNCs had no effect on proliferation. However, when A23187 was added to high dose (1:120) PHA-stimulated cord blood MNCs IFN-gamma production increased to levels comparable with adult PHA-stimulated MNCs. A similar pattern of response was seen when exogenous calcium chloride was added in place of A23187.

Induction of interferon-gamma by cord blood mononuclear cells is calcium dependent.

Human cord blood mononuclear cells (MNCs) are deficient in their ability to produce interferon-gamma (IFN-gamma). Previous studies have shown that phytohemagglutinin (PHA)-stimulated neonatal MNCs produced significantly less IFN-gamma than adult PHA-stimulated MNCs. The deficient IFN-gamma production is partly due to the absence of a macrophage-derived soluble mediator. Supernatants from PHA-stimulated adult macrophages and phorbol myristate acetate (PMA)-stimulated U937 cells (which were dialyzed prior to culture to remove PMA) increased IFN-gamma production in neonatal PHA-stimulated MNC (14 to 217 units/ml and 14 to 293 units/ml, respectively). The requirement for a soluble macrophage mediator was replaced by the addition of exogenous calcium chloride (CaCl2) to PHA-stimulated cord blood MNCs. The increase in IFN-gamma production by exogenous CaCl2 was blocked by the addition of the calcium channel blocker, manganese chloride (MnCl2). Furthermore, the increased IFN-gamma production by PHA-stimulated cord blood MNC in the presence of PHA-stimulated adult macrophage supernatant or PMA-stimulated U937 supernatant was abrogated by the addition of MnCl2, chlorpromazine, and verapamil. These data suggested that the soluble factor produced by PHA-stimulated adult macrophage supernatant and PHA-stimulated U937 supernatant induced IFN-gamma production in PHA-stimulated cord blood MNC by inducing calcium-dependent signals at more than one site.

Induction of class I major histocompatibility complex antigen expression by West Nile virus on gamma interferon-refractory early murine trophoblast cells.

Primary murine trophoblast giant cells (TGC) do not express detectable major histocompatibility complex (MHC) antigens and are refractory to the MHC-increasing effects of alpha and beta (virus-induced) interferons and gamma (immune type) interferon during early implantation (postcoital days 3.5-6). West Nile virus infection of primary TGC monolayers from postcoital-day-3.5 preimplantation blastocysts induced paternal MHC antigen expression within 16 hr, as detected by immunogold labeling for electron microscopy. Induction is unlikely to have been mediated by secreted virus-induced interferons or other factors, as it occurred in the presence of high concentrations of anti-alpha/beta interferon antibodies and was not induced by virus-inactivated supernatants from MHC-induced primary TGC cultures. Attempts to induce MHC antigen expression with poly(I.C) or recombinant tumor necrosis factor alpha in primary TGC cultures also failed. Thus, the apparent inhibition of MHC antigen expression in primary TGC during early implantation and their refractoriness to induction of de novo MHC antigen expression is not absolute. This may represent a maternal-and/or species-protective evolutionary device. As such, manipulation of this phenomenon may allow a conclusive assessment of the significance of inhibition of MHC antigen expression on trophoblast cells in the implanting semiallogeneic embryo.

Interferon-independent increases in class I major histocompatibility complex antigen expression follow flavivirus infection.

Infection of tertiary-passaged mouse embryo fibroblasts by four flaviviruses, West Nile (WNV), Kunjin, Murray Valley encephalitis and Japanese B encephalitis, resulted in a six- to 10-fold increase in the expression of individual H-2K and H-2D class I major histocompatibility complex (MHC) antigens 16 to 48 h after infection. The mechanism(s) by which flaviviruses increased antigen expression has not been fully elucidated, but appears to be mediated partly independently of interferon-beta (IFN-beta) secretion, as anti-IFN-alpha beta antibodies partially inhibited the WNV-induced increase but totally prevented increases caused by the addition of (i) pure IFN-beta, (ii) IFN-beta-containing supernatants from WNV-infected mouse embryo fibroblasts (MEF), or (iii) polyinosinic-polycytidylic acid. Actinomycin D treatment of MEF, which inhibited mRNA synthesis by greater than 90% as determined by [3H]uridine uptake, totally inhibited the increased MHC expression caused by WNV infection. Thus, the increase in class I MHC antigen expression following infection is dependent upon cellular RNA synthesis.

The secondary in vitro murine cytotoxic T cell response to the flavivirus, West Nile.

A secondary in vitro murine cytotoxic response to the flavivirus, West Nile (WNV) is described. Cytotoxic activity was obtained from spleen cells of mice primed 7 days previously with 10(6) p.f.u. WNV and boosted in vitro for a further 5 days with WNV-infected stimulator spleen cells. The cells responsible for lysis of WNV-infected target cells were restricted by class 1 H-2 antigens. The K region of the H-2k haplotype and both the K and D regions of the H-2d haplotype were permissive. The cytotoxic cells were virus-specific with respect to WNV and Influenza. The phenotype of the cells which mediated cytotoxicity was Thy 1+, Lyt 2+ and L3T4-; however an L3T4+ helper population was required for the optimal generation of the cytotoxic response in vitro.