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1.  Lentiviral expression system for the purification of secreted proteins from human cell cultures 
BMC Biotechnology  2016;16(1):66.
Recombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.
Using a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.
The procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.
PMCID: PMC5009704  PMID: 27590008
His-tag; Human cells; Lentiviral vector; Purification; Secreted proteins; Soluble CD4
2.  Safety of combination antiretroviral prophylaxis in high-risk HIV-exposed newborns: a retrospective review of the Canadian experience 
The optimal management of infants born to HIV-positive mothers who are untreated or have detectable viral load prior to delivery remains controversial. Despite the increasing use of combination antiretroviral therapy (cART) for post-exposure prophylaxis (PEP) of neonates at high risk of HIV infection, there is little safety and pharmacokinetic data to support this approach. The objective of this study was to evaluate the safety and tolerability of cART for PEP in HIV-exposed neonates.
Retrospective study on 148 cART and 145 Zidovudine (ZDV) monotherapy-exposed infants identified from four Canadian centres where cART for PEP has routinely been prescribed in high-risk situations. Physician-reported adverse events and clinical outcomes were extracted by chart review. Haematological and growth parameters at birth, one and six months of age were compared between cART and ZDV-exposed infants using multivariate mixed effects modelling.
Non-specific signs and symptoms were reported in 10.2% of cART recipients versus none of the ZDV recipients. Treatment was discontinued prematurely in 9.5% of cART recipients versus 2.1% of ZDV recipients (p=0.01). In the multivariate model, cART recipients had lower mean haemoglobin (decrease of 2.07 g/L) over the 6-month period compared with ZDV recipients (p=0.04), but no effect was seen on absolute neutrophil count. cART recipients had lower weight and smaller head circumference at birth and one month of age compared with ZDV-exposed infants; these differences were no longer significant at six months of age.
cART administered at treatment doses for PEP in neonates was generally well tolerated, though a higher incidence of non-specific signs and symptoms and early treatment discontinuation occurred among cART recipients.
PMCID: PMC4753845  PMID: 26880241
Prevention of mother-to-child transmission; HIV; neonatal prophylaxis; safety; combination antiretroviral therapy
3.  Early Initiation of Combination Antiretroviral Therapy in HIV-1–Infected Newborns Can Achieve Sustained Virologic Suppression With Low Frequency of CD4+ T Cells Carrying HIV in Peripheral Blood 
We studied a cohort of children initiated on treatment doses of combination antiretroviral therapy within 72 hours of birth. In children who achieved sustained virologic suppression, measures of HIV-1 reservoir size in peripheral blood were very low.
Background. A human immunodeficiency virus type 1 (HIV-1)–infected infant started on combination antiretroviral therapy (cART) at 30 hours of life was recently reported to have no detectable plasma viremia after discontinuing cART. The current study investigated the impact of early cART initiation on measures of HIV-1 reservoir size in HIV-1–infected children with sustained virologic suppression.
Methods. Children born to HIV-1–infected mothers and started on cART within 72 hours of birth at 3 Canadian centers were assessed. HIV serology, HIV-1–specific cell-mediated immune responses, plasma viremia, cell-associated HIV-1 DNA and RNA, presence of replication-competent HIV-1, and HLA genotype were determined for HIV-1–infected children with sustained virologic suppression.
Results. Of 136 cART-treated children, 12 were vertically infected (8.8%). In the 4 who achieved sustained virologic suppression, HIV serology, HIV-1–specific cell-mediated immune responses (Gag, Nef), and ultrasensitive viral load were negative. HIV-1 DNA was not detected in enriched CD4+ T cells of the 4 children (<2.6 copies/106 CD4+ T cells), whereas HIV-1 RNA was detected (19.5–130 copies/1.5 µg RNA). No virion-associated HIV-1 RNA was detected following mitogenic stimulation of peripheral blood CD4+ T cells (5.4–8.0 million CD4+ T cells) in these 4 children, but replication competent virus was detected by quantitative co-culture involving a higher number of cells in 1 of 2 children tested (0.1 infectious units/106 CD4+ T cells).
Conclusions. In perinatally HIV-1–infected newborns, initiation of cART within 72 hours of birth may significantly reduce the size of the HIV-1 reservoirs. Cessation of cART may be necessary to determine whether functional HIV cure can be achieved in such children.
PMCID: PMC4184383  PMID: 24917662
child; combination antiretroviral therapy; eradication; HIV; proviral DNA
4.  Quantiferon Gold-in-tube assay for TB screening in HIV infected children: influence of quantitative values 
BMC Infectious Diseases  2014;14:516.
HIV infected children are at increased risk of TB disease and require annual TB screening. Data on use of IGRA for TB screening in them are limited. We retrospectively evaluated the usefulness of Quantiferon Gold-in-tube test (QFT), an IGRA in screening for LTBI in relatively healthy, immunologically stable HIV infected children.
HIV infected children with no prior history of TB were screened for latent TB as part of routine care. They underwent risk of TB assessment, TST and QFT. QFT was repeated twice or three times depending on the quantitative values. Independent test validation was also performed.
Eighty one children had 109 QFT tests. All had adequate mitogen responses. The initial QFT was positive in 15 (18.5%) children; quantitative IGRA responses were 0.35-1.0 IU/mL in 9 (60%), 1.0-10 IU/mL in5 (33.3%) and >10 IU/mL in 1 (6.7%). None that tested positive had documented TB exposure or TB disease. Baseline characteristics in the QFT positive and negative groups were similar. Repeat testing within 17 weeks demonstrated reversion to negative in 79% of cases. Repeat blinded independent testing of all QFT positive results and a random selection of initial negative tests demonstrated concordance in 96% of cases. Seven children (QFT > 1.0 IU/mL or positive TST) were offered INH preventive therapy. In no case has TB disease developed in 2 years of close follow-up.
QFT is a valid method for LTBI screening relatively healthy, immunologically stable HIV infected children. However, reversion to negative on repeat testing and lack of correlation with TST results and risk of TB exposure makes interpretation difficult.
PMCID: PMC4181619  PMID: 25248406
HIV; Tuberculosis; Screening; IGRA; Quantiferon
6.  Risk factors and outcomes among children admitted to hospital with pandemic H1N1 influenza 
Limited data are available on disease characteristics and outcomes of children with 2009 pandemic influenza A(H1N1) virus infection (pandemic H1N1 influenza) who have required hospital admission.
We reviewed the charts of 58 children with pandemic H1N1 influenza admitted to a large pediatric hospital in Ontario, Canada, between May 8 and July 22, 2009. We compared risk factors, severity indicators and outcomes of these children with those of 200 children admitted with seasonal influenza A during the previous 5 years (2004/05 to 2008/09).
Children with pandemic H1N1 influenza were significantly older than those with seasonal influenza (median age 6.4 years v. 3.3 years). Forty-six (79%) of the children with pandemic H1N1 influenza had underlying medical conditions; of the other 12 who were previously healthy, 42% were under 2 years of age. Children admitted with pandemic H1N1 influenza were significantly more likely to have asthma than those with seasonal influenza (22% v. 6%). Two children had poorly controlled asthma, and 6 used inhaled medications only intermittently. The median length of stay in hospital was 4 days in both groups of children. Similar proportions of children required admission to the intensive care unit (21% of those with pandemic H1N1 influenza and 14% of those with seasonal influenza) and mechanical ventilation (12% and 10% respectively). None of the children admitted with pandemic H1N1 influenza died, as compared with 1 (0.4%) of those admitted with seasonal influenza.
Pandemic H1N1 influenza did not appear to cause more severe disease than seasonal influenza A. Asthma appears to be a significant risk factor for severe disease, with no clear relation to severity of asthma. This finding should influence strategies for vaccination and pre-emptive antiviral therapy.
PMCID: PMC2802603  PMID: 19926677
7.  A study of provider-caregiver communication in paediatric ambulatory care 
Paediatrics & Child Health  2006;11(4):217-221.
Provider-caregiver communication is a key ingredient in quality health care and patient safety, and effective communication has been shown to affect compliance and outcomes.
To identify and compare communication issues among three paediatric outpatient clinics.
In this prospective, qualitative study, a questionnaire was used to survey physicians, nurse practitioners and caregivers at three different infectious diseases clinics.
There was a statistically significant preponderance of families in the tuberculosis clinic for whom English was not the mother tongue and who were not fluent in English. Patients in the HIV clinic were less likely to be at their first appointment than were patients attending the other clinics. Patients in the general clinic were less likely to have been seen by the same physician on the previous visit. Parents from all three clinics were satisfied with the care they received, with communication and with rapport with their child. There was a trend toward parents in the tuberculosis clinic being happier with their clinic visit and less likely to complain about the wait time.
Language proficiency and lack of continuity of provider care were identified as potential risks for patient safety in the ambulatory setting. Further studies are necessary to identify language and cultural issues that may affect patient care in a tertiary paediatric hospital servicing a multiethnic population.
PMCID: PMC2528606  PMID: 19030275
Ambulatory care; Communication; Patient safety
8.  Response to a protease-inhibitor (ritonavir)-containing combination antiretroviral regimen in HIV-infected children 
The number of antiretroviral agents available for children who are failing existing therapy is limited. Data are lacking on the use of various combination regimens and the resulting viral load dynamics in such children.
Between March 1998 and March 2000, HIV-infected children younger than 18 years of age were studied in an open trial. The study regimen included ritonavir, with at least two drugs to which the virus was known or presumed to be sensitive. Subjects were ritonavir-naive and were included if they had high viral loads while receiving antiretroviral therapy. Patients had clinical assessments, CD4 counts and viral load monitoring.
Fifteen antiretroviral-experienced HIV-infected children were enrolled. Approximately 87% (13 of 15) had perinatally-acquired HIV; median age was 7.9 years (range 1.6 to 14.8). At enrolment, the median CD4 count was 557 cells/mm3 (range 57 to 1702) and the median viral load was 72,600 copies/mL (range 3626 to 796,440). The majority of children (73.3%) had increases in CD4 counts within 12 weeks. During this period, the median increase in CD4 counts over baseline was 30.0%. Approximately 73% (eight of 11) of subjects with initial improvements in CD4 counts had sustained increases at 32 to 48 weeks. Over the first 12 weeks, 60% (nine of 15) had greater than 0.5 log10 decreases in viral load. The improvement was sustained in 88.9% (eight of nine) of these patients at 32 to 48 weeks. Three patients discontinued therapy due to taste aversion.
Among pediatric patients with high viral loads while on existing therapy, the ritonavir-containing regimen was generally well tolerated. In a significant proportion of patients, modification of therapy was associated with sustained improvements in viral loads and CD4 counts over 32 to 48 weeks.
PMCID: PMC2094910  PMID: 18159430
Antiretroviral therapy; HIV infection; HIV viral load; Pediatrics; Protease inhibitor
9.  Quantification of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma by Using Small-Volume-Format Branched-DNA Assays 
Journal of Clinical Microbiology  1998;36(7):2096-2098.
We have developed small-volume (50 or 250 μl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.
PMCID: PMC104988  PMID: 9650972
10.  Pneumocystis prophylaxis for all, some, or no HIV-infected infants less than one year of age: A decision analysis approach 
Pneumocystis carinii pneumonia (PCP) is associated with significant mortality and morbidity among infants infected with human immunodeficiency virus (HIV). The preferred prophylaxis strategy for such infants is a subject of debate. Medical decision analysis was used to determine the preferred strategy for primary PCP prophylaxis among asymptomatic HIV-infected infants less than one year of age, and to determine the thresholds at which different variables influence decision making. Utility measures (health state preference values) were used to determine whether prophylaxis should be given to all, some or no infants. In this regard, some infants would receive prophylaxis if baseline CD4 counts are fewer than 1500 cells/mm3. The results suggest that the preferred option is to give prophylaxis to all asymptomatic HIV-infected infants despite CD4 counts, if the risk of PCP is equal to or greater than 25%. However, if the risk of PCP is less than 25%, prophylaxis is recommended for those infants with CD4 counts of fewer than 1500 cells/mm3. The results complement current guidelines regarding PCP prophylaxis for HIV-infected infants.
PMCID: PMC3250776  PMID: 22346469
Decision analysis; Pneumocystis; Prophylaxis
11.  Acute rheumatic fever: Findings of a hospital-based study and an overview of reported outbreaks 
To review the characteristics of reported outbreaks of acute rheumatic fever in the United States, and to determine if there is an increase in the incidence of acute rheumatic fever in the population served by the Hospital for Sick Children, Toronto, Ontario, the authors conducted a literature search and a retrospective review of inpatients and outpatients, satisfying the revised Jones criteria for the diagnosis of acute rheumatic fever, from 1972 to 1988. Patients satisfying the revised Jones criteria for the time period 1972–88 were included in the study. There have been eight articles reporting an increase in acute rheumatic fever in the United States. In three, the majority of children were white and from middle class suburban/rural communities in different geographic locations. Mucoid strains of group A streptococci were implicated but not confirmed as being associated with the outbreaks in three. The results of the chart review at the Hospital for Sick Children revealed that 83 cases satisfied the revised Jones criteria. The number of cases per 100,000 children (aged 18 years or less) per year, decreased progressively over the study period. Polyarthritis was the most frequently seen major criterion occurring in 73% of patients (61 of 83). The most frequently affected ethnic groups were Italians 23%, Afro-Canadians 19% and Orientals 8%. The reported outbreaks in the United States are multifocal and predominantly confined to white middle class children residing in suburban/rural communities. There was no evidence of an increase in the number of cases of acute rheumatic fever seen in the population served by the Hospital for Sick Children; there was a progressive decline in number of cases over the study period. The results facilitate the characterization of acute rheumatic fever within North America into three different patterns of occurrence.
PMCID: PMC3327974  PMID: 22553445
Acute rheumatic fever; Jones criteria; Outbreaks; Pharyngitis; Streptococcal infection
Journal of Clinical Investigation  1974;54(2):439-450.
The question of whether hypersensitivity to streptococcal antigens plays a role in the pathogenesis of the nonsuppurative sequelae of streptococcal infections remains at present unclear. As a first step in the approach to this question, the degree of cellular reactivity of peripheral blood leucocytes to streptococcal antigens was investigated in a number of rheumatic fever patients, patients with uncomplicated streptococcal infections, as well as normal healthy subjects.
Using the in vitro technique for the inhibition of capillary migration of peripheral blood leucocytes as an index of the degree of sensitivity to streptococcal antigens, the results indicate that patients with acute rheumatic fever exhibit an exaggerated cellular reactivity to these antigens and in particular to streptococcal cell membrane antigens. This abnormal response to streptococcal membrane antigens appears to persist in rheumatic subjects for at least 5 yr after the initial attack of rheumatic fever. Only Group A streptococcal membrane antigens elicited this unusual response in rheumatic subjects, since the cellular reactivity to Group C and D streptococcal membranes was the same in all groups. Patients with evidence of valvular disease exhibited the same degree of cellular reactivity to these antigens as did patients without clinical evidence of rheumatic heart disease.
The nature of the antigens responsible for the observed cellular response remains unknown. Enzymatic treatment of streptococcal cell walls and membranes designed to remove type-specific M proteins did not alter the observed cellular reactivity to the streptococcal antigens. The finding that an abnormal cellular response to certain streptococcal antigens is present only in rheumatic patients suggests that cell-mediated factors may play an important role in the disease process.
PMCID: PMC301572  PMID: 4603169

Results 1-12 (12)