Upregulation of human endogenous retrovirus-K (HML-2) mRNAs in hepatoblastoma: Identification of potential new immunotherapeutic targets and biomarkers.

PURPOSE: Hepatoblastoma is the most common liver malignancy in children. In order to advance therapy against hepatoblastoma, novel immunologic targets and biomarkers are needed. Our purpose in this investigation is to examine hepatoblastoma transcriptomes for the expression of a class of genomic elements known as Human Endogenous Retrovirus (HERVs). HERVs are abundant in the human genome and are biologically active elements that have been associated with multiple malignancies and proposed as immunologic targets in a subset of tumors. A sub-family of HERVs, HERV-K(HML-2) (HERV-K), have been shown to be tightly regulated in fetal development, making investigation of these elements in pediatric tumors paramount. METHODS: We first created a HERVK-FASTA file utilizing 91 previously described HML-2 proviruses. We then concatenated the file onto the GRCh38.95 cDNA library from Ensembl. We used this reference database to evaluate existing RNA-seq data from 10 hepatoblastoma tumors and 3 normal liver controls (GEO accession ID: GSE8977575). Quantification and differential proviral expression analysis between hepatoblastoma and normal liver controls was performed using the pseudo-alignment program Salmon and DESeq2, respectively. RESULTS: HERV-K mRNA was expressed in hepatoblastoma from multiple proviral loci. All expressed HERV-K proviral loci were upregulated in hepatoblastoma compared to normal liver controls. Five HERV-K proviruses (1q21.3, 3q27.2, 7q22.2, 12q24.33 and 17p13.1) were significantly differentially expressed (p-adjusted value <0.05, |log2 fold change| > 1.5) across conditions. The provirus at 17p13.1 had an approximately 300-fold increased expression in hepatoblastoma as compared to normal liver. This was in part due to the near absence of HERV-K mRNA at the 17p13.1 locus in fully differentiated liver samples. CONCLUSIONS: Our investigation demonstrates that HERV-K is expressed from multiple loci in hepatoblastoma and that the expression is increased for several proviruses compared to normal liver controls. Our results suggest that HERV-K mRNA expression may be useful as a biomarker in hepatoblastoma, given the large differential expression profiles in hepatoblastoma, with very low mRNA levels in liver control samples.

Human endogenous retrovirus-K mRNA expression and genomic alignment data in hepatoblastoma.

Human Endogenous Retroviruses are a class of genomic elements that are the result of ancient retroviral infection of the human germline. Many are biologically active elements that have been implicated in multiple diseases including cancer. The most recent class to invade the human genome is the HERV-K(HML-2) (HERV-K) family. Approximately 90 HERV-K proviruses and many smaller elements have been identified to date in the human genome. Additional proviruses are continually being discovered with the rapid advancement of deep-sequencing and long-read sequencing technologies. HERV-K proviruses are poorly annotated in human transcriptome databases making their analysis in RNA-seq data difficult. To enable analysis, we compiled the sequences of 91 HERV-K proviruses identified in NCBI GenBank (ID JN675007-JN675097) and created a proviral alignment tool for visualizing RNA-seq reads aligned across individual proviruses. This allowed us to analyse publicly available RNA-seq data from 10 hepatoblastoma samples and 3 normal liver controls (GEO Accession ID: GSE89775). This data report includes the raw FASTA sequence files of the HERV-K proviruses from NCBI, a differential gene expression list between hepatoblastoma samples, and genomic alignment figures from 5 HERV-K proviruses identified as differentially expressed in the companion research article "Upregulation of Human Endogenous Retrovirus-K (HML-2) mRNAs in hepatoblastoma: Identification of potential new immunotherapeutic targets and biomarkers [1]. The data provided here are available for other research groups interested in evaluating individual HERV-K proviral expression using RNA-seq data. Furthermore, the data analysis is highly flexible and will accommodate the addition of other HERV-K proviruses.

HIV-1 Rev interacts with HERV-K RcREs present in the human genome and promotes export of unspliced HERV-K proviral RNA.

BACKGROUND: The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. RESULTS: In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci (4p16.1b, 22q11.23 and most significantly 3q12.3) accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3' RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec. CONCLUSIONS: The HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes.

Ly49R activation receptor drives self-MHC-educated NK cell immunity against cytomegalovirus infection.

Natural killer (NK) cells mediate vital control of cancer and viral infection. They rely on MHC class I (MHC I)-specific self-receptors to identify and lyse diseased cells without harming self-MHC I-bearing host cells. NK cells bearing inhibitory self-receptors for host MHC I also undergo education, referred to as licensing, which causes them to become more responsive to stimulation via activation receptor signaling. Previous work has shown that licensed NK cells selectively expand during virus infections and they are associated with improved clinical response in human patients experiencing certain chronic virus infections, including HIV and hepatitis C virus. However, the importance of inhibitory self-receptors in NK-mediated virus immunity is debated as they also limit signals in NK cells emanating from virus-specific activation receptors. Using a mouse model of MHC I-dependent (H-2Dk) virus immunity, we discovered that NK cells depend on the Ly49G2 inhibitory self-receptor to mediate virus control, which coincided with host survival during murine cytomegalovirus infection. This antiviral effect further requires active signaling in NK cells via the Ly49R activation receptor that also binds H-2Dk. In tandem, these functionally discordant Ly49 self-receptors increase NK cell proliferation and effector activity during infection, resulting in selective up-regulation of CD25 and KLRG1 in virus-specific Ly49R+ Ly49G2+ NK cells. Our findings establish that paired self-receptors act as major determinants of NK cell-mediated virus sensing and immunity.

Evolution of the HIV-1 Rev Response Element during Natural Infection Reveals Nucleotide Changes That Correlate with Altered Structure and Increased Activity over Time.

The HIV-1 Rev response element (RRE) is a cis-acting RNA element characterized by multiple stem-loops. Binding and multimerization of the HIV Rev protein on the RRE promote the nucleocytoplasmic export of incompletely spliced mRNAs, an essential step in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies of lab-adapted HIV clones. However, in human infection, HIV evolves rapidly, and mechanistic studies of naturally occurring Rev and RRE sequences are essential to understanding this system. We previously described the functional activity of two RREs found in circulating viruses in a patient followed during the course of HIV infection. The early RRE was less functionally active than the late RRE, despite differing in sequence by only 4 nucleotides. In this study, we describe the sequence, function, and structural evolution of circulating RREs in this patient using plasma samples collected over 6 years of untreated infection. RRE sequence diversity varied over the course of infection, with evidence of selection pressure that led to sequence convergence as disease progressed being found. An increase in RRE functional activity was observed over time, and a key mutation was identified that correlates with a major conformational change in the RRE and increased functional activity. Additional mutations were found that may have contributed to increased activity as a result of greater Shannon entropy in RRE stem-loop II, which is key to primary Rev binding.IMPORTANCE HIV-1 replication requires interaction of the viral Rev protein with a cis-acting regulatory RNA, the Rev response element (RRE), whose sequence changes over time during infection within a single host. In this study, we show that the RRE is subject to selection pressure and that RREs from later time points in infection tend to have higher functional activity. Differences in RRE functional activity are attributable to specific changes in RNA structure. Our results suggest that RRE evolution during infection may be important for HIV pathogenesis and that efforts to develop therapies acting on this viral pathway should take this into account.

Characterization of APOBEC3 variation in a population of HIV-1 infected individuals in northern South Africa.

BACKGROUND: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. METHODS: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. RESULTS: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. CONCLUSIONS: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .

Next generation sequencing reveals a high frequency of CXCR4 utilizing viruses in HIV-1 chronically infected drug experienced individuals in South Africa.

BACKGROUND: Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses). In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. The use of Maraviroc is reserved for salvage therapy in South Africa. OBJECTIVE: In this study, we examined the frequency of R5 and X4 viruses, using next generation sequencing, in patients under treatment to draw inferences on the utility of Maraviroc in a South African population. STUDY DESIGN: Proviral DNA was isolated from peripheral blood mononuclear cells (PBMC) of 72 chronically HIV infected patients on antiretroviral treatment. HIV V3 loop gene was amplified and sequenced on an Illumina MiniSeq platform. Viral subtypes were determined by the jumping profile Hidden Markov Model (jpHMM) and REGA genotyping tools. De Novo consensus sequences were derived for the majority and minority populations for each patient using Geneious® software version 8.1.5. HIV-1 tropism was inferred using PSSMsinsi, Geno2pheno and Phenoseq-C web-based tools. RESULTS: Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients exclusively harboured R5 viral quasispecies; and 21% (15/72) exclusively harbored X4 viral quasispecies. Twenty five percent of patients (18/72) harbored dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) harbored the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) harbored the R5X4+ mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harbored X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C harboured X4 viruses (χ2 = 3.58; p = .058), and 57% of these (13/23) harbored X4 viruses exclusively. CD4+ cell count less than 350 cell/µl was associated with the presence of X4 viruses (χ2 = 4.99; p = .008). CONCLUSION: The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viruses.

Effect of intercalator and Lewis acid-base branched peptide complex formation: boosting affinity towards HIV-1 RRE RNA.

High throughput screening of a 4096 compound library of boronic acid and acridine containing branched peptides revealed compounds that have dissociation constants in the low nanomolar regime for HIV-1 RRE IIB RNA. We demonstrate that branched peptide boronic acids A5, A6, and A7 inhibit the production of p24, an HIV-1 capsid protein, in a dose-dependent manner.

Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding.

Limited nucleotide changes in the Rev response element (RRE) during HIV-1 infection alter overall Rev-RRE activity and Rev multimerization.

HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.

A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes.

BACKGROUND: Reports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates. METHODS: Pvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples. RESULTS: Genotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele. CONCLUSIONS: The LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.

Depression in children and adolescents two months after the death of a parent.

BACKGROUND: This study examined depressive symptoms in bereaved children and adolescents two months after the death of a parent. METHODS: Participants were 325 children and adolescents bereaved of a parent approximately two months prior to the study. They were compared to 129 non-bereaved community controls and 110 non-bereaved depressed controls. Participants and their parents were interviewed regarding the child's depressive symptoms. Possible moderating factors for depression in bereaved children were examined. RESULTS: 25% of the bereaved participants experienced a major depressive episode (MDE) compared to 1% of the community controls. An additional 24% of the bereaved participants experienced a sub-syndromal depressive episode, defined as 3 or 4 depressive symptoms, compared to 4% of the community controls. Factors correlated with occurrence of MDE in the bereaved children in exploratory analyses were (1) history of MDE in the child and (2) history of alcoholism in a parent. Guilt/worthlessness, psychomotor disturbance, and low energy in the context of an MDE predicted membership in the depressed control group over the bereaved group. LIMITATIONS: The relationship between an MDE in the bereaved child and parent history of alcoholism is exploratory, as the p-value for this correlation was greater than the α adjusted for multiple comparisons. The bereaved child's history of MDE was based on the child's and parent's memories of depressive symptoms. CONCLUSIONS: The death of a parent is a risk factor for depressive symptoms and depressive episodes in children and adolescents two months after the death.

Arbovirus prevalence in mosquitoes, Kenya.

Few studies have investigated the many mosquito species that harbor arboviruses in Kenya. During the 2006-2007 Rift Valley fever outbreak in North Eastern Province, Kenya, exophilic mosquitoes were collected from homesteads within 2 affected areas: Gumarey (rural) and Sogan-Godud (urban). Mosquitoes (n = 920) were pooled by trap location and tested for Rift Valley fever virus and West Nile virus. The most common mosquitoes trapped belonged to the genus Culex (75%). Of 105 mosquito pools tested, 22% were positive for Rift Valley fever virus, 18% were positive for West Nile virus, and 3% were positive for both. Estimated mosquito minimum infection rates did not differ between locations. Our data demonstrate the local abundance of mosquitoes that could propagate arboviral infections in Kenya and the high prevalence of vector arbovirus positivity during a Rift Valley fever outbreak.

Molecular-based assay for simultaneous detection of four Plasmodium spp. and Wuchereria bancrofti infections.

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)-based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction-fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85-93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.

Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people.

Malaria therapy, experimental, and epidemiological studies have shown that erythrocyte Duffy blood group-negative people, largely of African ancestry, are resistant to erythrocyte Plasmodium vivax infection. These findings established a paradigm that the Duffy antigen is required for P. vivax erythrocyte invasion. P. vivax is endemic in Madagascar, where admixture of Duffy-negative and Duffy-positive populations of diverse ethnic backgrounds has occurred over 2 millennia. There, we investigated susceptibility to P. vivax blood-stage infection and disease in association with Duffy blood group polymorphism. Duffy blood group genotyping identified 72% Duffy-negative individuals (FY*B(ES)/*B(ES)) in community surveys conducted at eight sentinel sites. Flow cytometry and adsorption-elution results confirmed the absence of Duffy antigen expression on Duffy-negative erythrocytes. P. vivax PCR positivity was observed in 8.8% (42/476) of asymptomatic Duffy-negative people. Clinical vivax malaria was identified in Duffy-negative subjects with nine P. vivax monoinfections and eight mixed Plasmodium species infections that included P. vivax (4.9 and 4.4% of 183 participants, respectively). Microscopy examination of blood smears confirmed blood-stage development of P. vivax, including gametocytes. Genotyping of polymorphic surface and microsatellite markers suggested that multiple P. vivax strains were infecting Duffy-negative people. In Madagascar, P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. Further studies are necessary to identify the parasite and host molecules that enable this Duffy-independent P. vivax invasion of human erythrocytes.

Differential patterns of infection and disease with P. falciparum and P. vivax in young Papua New Guinean children.

BACKGROUND: Where P. vivax and P. falciparum occur in the same population, the peak burden of P. vivax infection and illness is often concentrated in younger age groups. Experiences from malaria therapy patients indicate that immunity is acquired faster to P. vivax than to P. falciparum challenge. There is however little prospective data on the comparative risk of infection and disease from both species in young children living in co-endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: A cohort of 264 Papua New Guinean children aged 1-3 years (at enrolment) were actively followed-up for Plasmodium infection and febrile illness for 16 months. Infection status was determined by light microscopy and PCR every 8 weeks and at each febrile episode. A generalised estimating equation (GEE) approach was used to analyse both prevalence of infection and incidence of clinical episodes. A more pronounced rise in prevalence of P. falciparum compared to P. vivax infection was evident with increasing age. Although the overall incidence of clinical episodes was comparable (P. falciparum: 2.56, P. vivax 2.46 episodes / child / yr), P. falciparum and P. vivax infectious episodes showed strong but opposing age trends: P. falciparum incidence increased until the age of 30 months with little change thereafter, but incidence of P. vivax decreased significantly with age throughout the entire age range. For P. falciparum, both prevalence and incidence of P. falciparum showed marked seasonality, whereas only P. vivax incidence but not prevalence decreased in the dry season. CONCLUSIONS/SIGNIFICANCE: Under high, perennial exposure, children in PNG begin acquiring significant clinical immunity, characterized by an increasing ability to control parasite densities below the pyrogenic threshold to P. vivax, but not to P. falciparum, in the 2(nd) and 3(rd) year of life. The ability to relapse from long-lasting liver-stages restricts the seasonal variation in prevalence of P. vivax infections.

Assessment of depression in children and adolescents.

Depression assessment instruments are valuable tools in the treatment of children and adolescents. Available instruments include diagnostic interviews, self-administered rating scales, and observer-rated scales. To select an appropriate instrument, the user must define the goal of the assessment and then identify instruments with the properties that match this goal. This article discusses how to choose an assessment instrument and gives an overview of currently available depression assessment instruments. Important considerations include how and by whom an instrument is administered, what kind of data are obtained by the instrument, and the validity and reliability of the instrument. Standardized instruments can greatly improve the assessment process, but the user must not overinterpret or misinterpret the results.

HIV-1 variants from a perinatal transmission pair demonstrate similar genetic and replicative properties in tonsillar tissues and peripheral blood mononuclear cells.

Human immunodeficiency virus type 1 (HIV-1) can be acquired through oropharyngeal tissues in breastfeeding infants. Efforts to better understand the determinants of breast milk transmission are hampered by the lack of a relevant oral human mucosa model and well-defined breast milk-derived viruses. This study used human ex vivo palatine tonsil tissues and peripheral blood mononuclear cells (PBMCs) to characterize the genetic, biological, and replicative properties of HIV-1 variants obtained from a perinatal transmission pair. Unique viral populations from maternal breast milk and infant blood were identified by gp120 V1-V2- and V3-specific heteroduplex tracking assays (HTAs). Full-length infectious recombinant viruses, containing a common HIV-1 NL4-3 genetic background, were generated with V1-V3 gp120 fragments from maternal and infant isolates representing the major viral populations identified in the HTAs. The resulting recombinant viruses used the CCR5 coreceptor, were nonsyncytium forming, and demonstrated replication properties similar to those of parental and control viruses in PBMCs and tonsillar explants. These findings indicate that viruses from breast milk cells and infant blood can infect PBMCs and tonsil tissues. The maternal and infant HIV-1 viruses detailed here will provide useful tools for defining the viral and host factors that contribute to HIV breastfeeding transmission.

Salivary secretory leukocyte protease inhibitor and oral candidiasis in human immunodeficiency virus type 1-infected persons.

Oropharyngeal candidiasis, typically caused by Candida albicans, is the most common oral disease associated with human immunodeficiency virus type 1 (HIV-1) infection. Secretory leukocyte protease inhibitor (SLPI), a 12-kDa antiprotease, suppresses the growth of C. albicans in vitro. To determine whether the mucosal protein plays a role in protecting oral tissues against fungal infection, we conducted a cross-sectional study investigating the oral and systemic health and salivary SLPI levels in 91 dentate HIV-1-infected adults receiving medical care in the southeastern United States. Participants with a self-reported history of clinical oropharyngeal candidiasis during the previous 2 years constituted the test group (n = 52), while the comparison group (n = 39) had no oropharyngeal candidiasis during that period. Data collected from medical records, oral examination, and SLPI enzyme-linked immunosorbent assay quantitation of whole saliva were analyzed by t test, analysis of variance, linear regression, and unconditional logistic regression. The test group had a significantly higher mean salivary SLPI level than the comparison group (1.9 microg/ml versus 1.1 microg/ml, P < 0.05). Linear regression modeling identified CD4 cell count and history of oropharyngeal candidiasis as key predictors of salivary SLPI and revealed a significant interaction (P < 0.05) between immunosuppression (CD4 cell count below 200 cells/ microl) and positive history of oropharyngeal candidiasis in predicting salivary SLPI level. By logistic regression modeling, a salivary SLPI level exceeding 2.1 microg/ml, low CD4 count, antiretroviral monotherapy, and smoking were key predictors of oropharyngeal candidiasis. These data support a key role for SLPI in the oral mucosal defense against C. albicans. The antimicrobial mucosal protein may serve as an indicator of previous oropharyngeal candidiasis infection among immunosuppressed persons.