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A 7-year-old boy presented to the emergency department with fever, cough, congestion, abdominal pain, myalgias, and morbilliform rash. Several aspects of the patient’s history, including recent travel, living on a farm, exposure to sick contacts, and new medications, resulted in a wide differential diagnosis. Initial laboratory testing revealed leukocytosis with neutrophilia and elevated atypical lymphocytes, but did not reveal any infectious causes of illness. He was discharged from the hospital, but then represented to the emergency department a day later with worsening rash, continued fever, abdominal pain, and poor intake. He was then admitted. A more comprehensive laboratory evaluation was initiated. During this hospital course, the patient’s physical examination changed when he developed head and neck edema, and certain laboratory trends became clearer. With the assistance of several specialists, the team was able to reach a more definitive diagnosis and initiate treatment to appropriately manage his condition.

Many mechanisms responsible for COVID-19 pathogenesis are well-established, but COVID-19 includes features with unclear pathogenesis, such as autonomic dysregulation, coagulopathies, and high levels of inflammation. The receptor for SARS-CoV-2 spike protein’s receptor binding domain (RBD) is angiotensin converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop antibodies that have negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity – ACE2-like abzymes. To explore this hypothesis, we studied patients hospitalized with COVID-19 who had plasma samples available obtained about 7 days after admission. ACE2 is a metalloprotease that requires Zn2+for activity. However, we found that the plasma from some patients studied could cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using disodium ethylenediaminetetraacetate (EDTA) anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+was added or the plasma was diluted to decrease EDTA concentration. After processing samples by 100 kDa size exclusion columns and protein A/G adsorption, which depleted immunoglobulin by >99.99%, the plasma samples did not cleave the ACE2 substrate peptide. The data suggest that some patients with COVID-19 develop antibodies with abzyme-like activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate many physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure COVID-19 pathogenesis.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Beyond their pulmonary disease, many COVID-19 patients experience a complex constellation of characteristics, including hyperinflammatory responses, autoimmune disorders, and coagulopathies. However, the pathogenesis of these aspects of COVID-19 is obscure. More than 90% of people are latently infected with the lymphotropic herpesviruses Epstein–Barr Virus (EBV) and/or Human Herpesvirus-6 (HHV-6). Some of the inflammatory features of COVID-19 resemble clinical syndromes seen during EBV and HHV-6 infection, and these latent viruses can be reactivated by inflammatory mediators. We hypothesized that EBV and HHV-6 reactivation might be a common feature of early COVID-19, particularly in patients with more inflammation. We tested for EBV and HHV-6 reactivation in 67 patients acutely hospitalized with COVID-19 using previously validated quantitative PCR assays on the plasma. In our cohort, we found that 15/67 (22.4%) patients had detectable EBV and 3/67 (4.5%) had detectable HHV-6. This frequency of activation is somewhat more than the frequency reported for some healthy cohorts, such as blood donors and other healthy control cohorts. There was no association between EBV or HHV-6 and markers indicative of more inflammatory disease. We conclude that EBV and HHV-6 activation at about day 7 of hospitalization occurred in a modest fraction of our cohort of COVID-19 patients and was not associated with high levels of inflammation. In the modest fraction of patients, EBV and HHV-6 reactivation could contribute to some features of acute disease and pre-disposition to post-acute sequelae in a subset of patients.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated

* Abbreviations: COVID-19 — : coronavirus disease 2019 nAb — : neutralizing antibody SARS-CoV-2 — : severe acute respiratory syndrome coronavirus 2 After having coronavirus disease 2019 (COVID-19), patients develop a humoral immune response thought to protect against reinfection, but antibody levels can decline over time. Understanding how long antibody levels remain high enough to prevent infection is important in understanding, absent vaccination, whether children may be vulnerable to COVID-19 and in modeling how COVID-19 spreads through the population. The levels of antibodies capable of neutralizing the virus (neutralizing antibodies [nAbs]) provide a direct marker of a protective humoral immune response. The nAbs can be measured directly by determining how well a patient’s serum inactivates virus when a known amount of virus is placed on cells in tissue culture, which is known as a plaque reduction neutralization test. The remarkably safe and effective approved COVID-19 vaccines elicit potent antibody responses against portions of the virus mediating attachment to host cells and viral entry, the spike protein and its receptor-binding domain. The concentration of antibodies a patient has against spike … Address correspondence to: Andrea T. Cruz, Department of Pediatrics, Baylor College of Medicine, 6621 Fannin St, Suite A2210, Houston, TX 77030. E-mail: acruz{at}bcm.edu

Multisystem inflammatory syndrome in children (MIS-C) is a serious, sometimes life-threatening late complication of coronavirus disease 2019 (COVID-19) with multiorgan involvement and evidence of immune activation. The pathogenesis of MIS-C is not known, nor is the pathogenesis of the severe organ damage that is the hallmark of MIS-C. Human herpesvirus 6 (HHV-6), the virus responsible for roseola, is a ubiquitous herpesvirus that causes close to universal infection by the age of 3 years. HHV-6 remains latent for life and can be activated during inflammatory states, by other viruses, and by host cell apoptosis. HHV-6 has been associated with end-organ diseases, including hepatitis, carditis, and encephalitis. In addition, ∼1% of people have inherited chromosomally integrated human herpesvirus 6 (iciHHV-6), which is HHV-6 that has been integrated into chromosomal telomeric regions and is transmitted through the germ line. iciHHV-6 can be reactivated and has been associated with altered immune responses. We report here a case of MIS-C in which an initial high HHV-6 DNA polymerase chain reaction viral load assay prompted testing for iciHHV-6, which yielded a positive result. Additional research may be warranted to determine if iciHHV-6 is commonly observed in patients with MIS-C and, if so, whether it may play a part in MIS-C pathogenesis.

Significance We report a vaccine platform to express vaccine antigens on the surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrate the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface of Escherichia coli to produce killed whole-cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates interferon-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly protective coronavirus vaccine since a betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against alphacoronavirus porcine epidemic diarrhea virus. When using a vaccine-appropriate bacteria vector, this inexpensive vaccine platform offers the potential for use in developing countries., As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reducedE. colito express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.Significance StatementWe report a new vaccine platform to express vaccine antigens on surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrated the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface ofE.colito produce killed whole cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates IFN-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly-protective coronavirus vaccine since a Betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against Alphacoronavirus PEDV. When using a vaccine appropriate bacteria vector, this inexpensive new vaccine platform offers the potential for use in developing countries.

* Abbreviations: COVID-19 — : coronavirus disease 2019 MIS-C — : multisystem inflammatory syndrome in children RBD — : receptor-binding domain S — : severe acute respiratory syndrome coronavirus 2 spike protein SARS-Co-V-2 — : severe acute respiratory syndrome coronavirus 2 Coronavirus disease 2019 (COVID-19) is arguably the most socially and economically disruptive pandemic since the 1918 influenza pandemic. Although pediatric COVID-19 shares features with the adult disease, there are several differences. Children produce virus in amounts at least equal to adults, if not higher,1 and can transmit the virus, just as adults can.2 School-aged children are generally less-severely affected than infants or adults, but some children without significant underlying disease become ill or die in a disease process analogous to the one most commonly seen in adults: severe pulmonary disease and respiratory failure.3–6 Children and adults appear to have different humoral immune responses to COVID-19.7 A small fraction of children with COVID-19 experience a hyperinflammatory process,8–13 termed multisystem inflammatory syndrome in children (MIS-C) in the United States, with features distinct from Kawasaki disease.14 This case definition includes ≥2 of these symptoms: rash, conjunctivitis, or mucocutaneous inflammation; hypotension; cardiac disease; coagulopathy; or acute gastrointestinal problems.15 MIS-C, which is not correlated with viral load levels, typically appears some time after initial infection.13 MIS-C’s incidence is difficult to determine, given the high rate of asymptomatic infection.12 Adults with COVID-19 also can experience inflammatory disorders: coagulopathies, vasculitis, cardiomyositis, and neuroinflammatory processes.16–18 That the most effective therapy to reduce mortality in adults with severe COVID-19 yet established is an immune suppressant, dexamethasone,19 reveals the importance of inflammatory and immune-mediated pathologies. In this issue of … Address correspondence to Steven Zeichner, MD, PhD, Department of Pediatrics, University of Virginia, Box 801349, Charlottesville, VA 22908. E-mail: zeichner{at}virginia.edu

* Abbreviation: COVID-19 — : coronavirus disease of 2019 The impact of the coronavirus disease of 2019 (COVID-19), which is caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2,1 has been widespread, with >500 000 cases diagnosed in >170 countries since the virus was identified in January 2020.2 The preliminary data have been focused on severe respiratory manifestations, which are seen predominantly in adults, with scant initial data on the burden of COVID-19 in children.3 We, therefore, read with interest the findings of Dong et al,4 who reported in this issue of Pediatrics a series of >2000 children with suspected or confirmed COVID-19. The authors found that 4% of virologically confirmed cases had asymptomatic infection, and this rate almost certainly understates the true rate of asymptomatic infection because many children who are asymptomatic are unlikely to be tested. Among children who were symptomatic, 5% had dyspnea or hypoxemia (a substantially lower percentage than what has been reported for adults3), and 0.6% progressed to acute respiratory distress syndrome or multiorgan system dysfunction (a rate that is also lower than that seen in adults). Preschool-aged children and infants were more likely than older children to have severe clinical manifestations. There are several salient points … Address correspondence to Andrea T. Cruz, MD, MPH, Department of Pediatrics, Baylor College of Medicine, 6621 Fannin St, Suite A2210, Houston, TX 77030. E-mail: acruz{at}bcm.edu

A 16-year-old girl presented to the emergency department with intermittent fevers and worsening abdominal pain of 5 weeks duration. She had a history of travel to a less developed country and exposure to possible infectious diseases. Abdominal imaging and blood tests revealed diffuse mesenteric lymphadenopathy, elevated transaminases, and elevation of inflammatory markers. Gastroesophageal and colon endoscopies revealed gastric ulcers, and the patient was discharged with a presumptive diagnosis of systemic juvenile idiopathic arthritis given the lymphadenopathy seen on imaging, serositis, sacroiliac joint stiffness noted on physical examination, and pain relief with celecoxib. She presented again 4 days later with worsening abdominal tenderness, elevated transaminases, and new-onset abdominal distention. Tissue biopsy yielded the diagnosis and directed appropriate treatment.

Background/objectives Atopic dermatitis (AD) is a common chronic inflammatory skin condition associated with high transepidermal water loss, high skin pH, and Staphylococcus aureus skin colonization. The treatment of AD with bath additives remains highly debated. Recent evidence suggests that dilute apple cider vinegar (ACV) may improve skin barrier integrity in AD, but its safety and efficacy are not well studied. This pilot split-arm study analyzed the effect of dilute apple cider vinegar soaks on skin barrier integrity in patients with atopic dermatitis as measured by skin transepidermal water loss and skin pH. Methods A total of 22 subjects (11 AD and 11 healthy controls) were enrolled. Subjects soaked both of their forearms for 14 days, with one arm in dilute ACV (0.5% acetic acid) and the other in water 10 minutes daily. Transepidermal water loss and pH were measured pre- and post-treatment. Results In both groups, transepidermal water loss increased and pH decreased at 0 minutes post-ACV treatment, but these effects were not sustained at 60 minutes. In total, 72.7% (16/22) of subjects reported mild side effects from ACV with improvement after discontinuing the soaks. Conclusions Dilute ACV soaks have no significant effect on skin barrier integrity but caused skin irritation in a majority of subjects. Study limitations include analysis of a single brand, dilution, and application of ACV. Future studies are needed to explore whether lower concentrations of ACV soaks or other applications such as a leave-on acidic ointment could improve skin barrier integrity in a safe, nonirritating way.

No effective therapy to eliminate the HIV latently infected cell reservoir has been developed. One approach, “shock and kill”, employs agents that activate HIV, subsequently killing the activated infected cells and/or virus. Shock and kill requires agents that safely and effectively activate HIV. One class of activation agents works through classical NF-κB pathways, but global NF-κB activators are non-specific and toxic. There exist two major IκBs: IκBα, and IκBε, which hold activating NF-κB subunits in the cytoplasm, releasing them for nuclear transit upon cell stimulation. IκBα was considered the main IκB responsible for gene expression regulation, including HIV activation. IκBε is expressed in cells constituting much of the latent HIV reservoir, and IκBε knockout mice have a minimal phenotype, suggesting that IκBε could be a valuable target for HIV activation and reservoir depletion. We previously showed that targeting IκBε yields substantial increases in HIV expression. Here, we show that IκBε holds c-Rel and p65 activating NF-κB subunits in the cytoplasm, and that targeting IκBε with siRNA produces a strong increase in HIV expression associated with enhanced c-Rel and p65 transit to the nucleus and binding to the HIV LTR of the activating NF-κBs, demonstrating a mechanism through which targeting IκBε increases HIV expression. The findings suggest that it may be helpful to develop HIV activation approaches, acting specifically to target IκBε and its interactions with the NF-κBs.

Short bowel syndrome (SBS) presents an increasing problem in pediatrics. SBS often results from surgical resection of necrotic bowel following necrotizing enterocolitis or treatment of anatomic gastrointestinal defects. SBS is associated with significant morbidity and mortality, and creates substantial burdens for patients, families, and the health system. Recent reports have demonstrated that the fecal microbiome of children with SBS is significantly different from healthy control and severe intestinal microbial imbalances is associated with poor growth. We hypothesized that children with SBS and adverse clinical features such as PN dependent, shorter bowel length and lack of ileocecal valve would demonstrate more gut dysbiosis compare with the SBS non-PN dependent. An improved understanding of SBS pathogenesis would enhance management and potentially suggest new interventions. We studied microbial communities of SBS and control non-SBS patients from the jejunum, obtained endoscopically or by ostomy aspiration, and stool. We enrolled SBS patients who did and did not require parenteral nutrition (PN), as a surrogate marker for the seriousness of their disease. We studied the microbiota using high-throughput DNA sequencing of 16S rRNA genes and statistical analyses. We found that microbial diversity was significantly greater in jejunal aspirate than in stool samples in SBS patients, unlike non-SBS patients; that SBS patients receiving enteral feeds had greater diversity, and that SBS patients on PN and enteral feeds had lower differences in diversity in jejunal vs. stool samples. We found a trend toward increased diversity in patients with an intact ileocecal valve, and found that certain taxa were more abundant in the certain sample types, and in SBS patients vs. non-SBS patients. SBS patients have lower microbial diversity, especially patients with more severe disease, patients requiring PN, and those lacking an ileocecal valve. SBS patients, particularly those with more complex characteristics, exhibit differences in their intestinal microbiota. Particular individual taxa were over- and under-represented in patients with more unfavorable disease. While diminished diversity and alterations in microbiota composition are likely consequences of SBS, future efforts aimed at increasing microbial diversity and interventions targeting specific microbiota characteristics might constitute a testable approach to ameliorate some clinical SBS clinical consequences.

Introduction Atopic dermatitis is a common skin disease characterized by altered cutaneous immunity in which patients often exhibit lower skin microbiota diversity compared to healthy skin and are prone to colonization by Staphylococcus aureus. Apple cider vinegar has been shown to have antibacterial effects; however, its effects on the skin microbiome have not previously been well-described. Objectives We aimed to examine the effects of topical dilute apple cider vinegar soaks on Staphylococcus aureus abundance, skin bacterial microbiome composition, and skin bacterial microbiome diversity in atopic dermatitis participants compared to healthy skin. Methods Eleven subjects with atopic dermatitis and 11 healthy controls were enrolled in this randomized, non-blinded, single-institution, split-arm pilot study. Subjects soaked one forearm in dilute apple cider vinegar (0.5% acetic acid) and the other forearm in tap water for 10 minutes daily. Skin bacteria samples were collected from subjects’ volar forearms before and after 14 days of treatment. 16S sequencing was used to analyze Staphylococcus aureus abundance and skin bacterial microbiome composition, and alpha diversity of microbiota were determined using Shannon diversity index. Results There was no difference in skin bacterial microbiome in atopic dermatitis subjects after 2 weeks of daily water or apple cider vinegar treatments (p = 0.056 and p = 0.22, respectively), or in mean abundance of S. aureus on apple cider vinegar-treated forearms (p = 0.60). At 2 weeks, the skin bacterial microbiomes of healthy control subjects were not significantly different from the skin bacterial microbiome of atopic dermatitis subjects (p = 0.14, 0.21, 0.12, and 0.05). Conclusions Our results suggest that daily soaks in 0.5% apple cider vinegar are not an effective method of altering the skin bacterial microbiome in atopic dermatitis. Further studies are needed to explore the effects of different concentrations of apple cider vinegar on skin microflora and disease severity. Trial number UVA IRB-HSR #19906.

Regulation of expression of HTLV-1 gene products from integrated proviruses plays an important role in HTLV-1-associated disease pathogenesis. Previous studies have shown that T cell receptor (TCR)- and phorbol ester (PMA) stimulation of chronically infected CD4 T cells increases the expression of integrated HTLV-1 proviruses in latently infected cells, however the mechanism remains unknown. Analysis of HTLV-1 RNA and protein species following PMA treatment of the latently HTLV-1-infected, FS and SP T cell lines demonstrated rapid induction of tax/rex mRNA. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability while the stability of unspliced or singly spliced HTLV-1 RNAs did not increase. Tax/rex mRNA in the HTLV-1 constitutively expressing cell lines exhibited high basal stability even without PMA treatment. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression at least in part through increased tax/rex mRNA stability.

Virologic response to highly active antiretroviral therapy (HAART) treatment of human immunodeficiency virus (HIV) infection depends on viral sensitivity to antiretrovirals (ARV’s) and excellent medication adherence. Adolescents with vertically-acquired HIV may require complicated regimens due to significant treatment experience and often have poor medication adherence. A retrospective chart review identified five adolescents with vertically acquired HIV and plasma HIV viral load rebound or nonresponse on a stable HAART regimen followed by a period of directly observed therapy (DOT) in a clinic or hospital setting with serial viral load measurements. Four subjects had a virologic response (mean decline 1.15 log) after DOT. A response to HAART can be seen despite ARV resistance using DOT, and treatment-experienced patients seemingly unresponsive to HAART may be non-adherent even with reassuring adherence measures. A period of clinic-monitored DOT may allow diagnosis of non-adherence, discussion of medication barriers and avoidance of unnecessary medication changes.

We examined 28 children with HIV-1 infection who were not responding to existing antiviral regimens and were enrolled into clinical trials conducted at the National Cancer Institute to receive salvage therapy. In 3 of the 28 patients (10.7%), the Q151M complex amino acid substitutions were identified. The three patients had received nucleoside reverse transcriptase inhibitor (NRTI) monotherapy and/or combination regimens with multiple NRTIs for 4.3-8.6 years prior to the study. Recombinant infectious clones generated by incorporating the RT-encoding region of HIV-1 isolated from patients' plasma samples were highly resistant to zidovudine, didanosine and stavudine, while they were moderately resistant to lamivudine and tenofovir disoproxil fumarate (TDF). TDF-containing regimens reduced HIV-1 viremia in two of the three children carrying the Q151M complex. These data suggest that the Q151M could be prevalent in pediatric patients with long-term NRTI monotherapy and/or dual NRTI regimens and that HAART regimens containing TDF may be meritorious in such patients.

Background Despite much work, safe and effective approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1 remain an elusive goal. Patients infected with HIV-1 treated with cytotoxic agents or bone marrow transplantation can experience decreases in the reservoir of HIV-1 latently infected cells. Other viruses capable of long-term latency, such as herpesviruses, can sense host cell apoptosis and respond by initiating replication. These observations suggest that other viruses capable of long-term latency, like HIV-1, might also sense when its host cell is about to undergo apoptosis and respond by initiating replication. Results Pro-monocytic (U1) and lymphoid (ACH-2) HIV-1 persistently infected cell lines were treated with cytotoxic drugs – doxorubicin, etoposide, fludarabine phosphate, or vincristine – and activation of latent HIV-1 was evaluated using assays for HIV-1 RNA and p24 production. Both cell lines showed dose-dependent increases in apoptosis and associated HIV-1 activation following exposure to the cytotoxic agents. Pretreatment of the cells with the pan-caspase inhibitor Z-VAD-FMK prior to exposure to the cytotoxic agents inhibited apoptosis and viral activation. Direct exposure of the latently infected cell lines to activated caspases also induced viral replication. HIV-1 virions produced in association with host cell apoptosis were infectious. Conclusions The results indicate that latent HIV-1 can sense when its host cell is undergoing apoptosis and responds by completing its replication cycle. The results may help explain why patients treated with cytotoxic regimens for bone marrow transplantation showed reductions in the reservoir of latently infected cells. The results also suggest that the mechanisms that HIV-1 uses to sense and respond to host cell apoptosis signals may represent helpful new targets for approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1.

The oral microbial community (microbiota) plays a critical role in human health and disease. Alterations in the oral microbiota may be associated with disorders such as gingivitis, periodontitis, childhood caries, alveolar osteitis, oral candidiasis and endodontic infections. In the immunosuppressed population, the spectrum of potential oral disease is even broader, encompassing candidiasis, necrotizing gingivitis, parotid gland enlargement, Kaposi’s sarcoma, oral warts and other diseases. Here, we used 454 pyrosequencing of bacterial 16S rRNA genes to examine the oral microbiome of saliva, mucosal and tooth samples from HIV-positive and negative children. Patient demographics and clinical characteristics were collected from a cross-section of patients undergoing routine dental care. Multiple specimens from different sampling sites in the mouth were collected for each patient. The goal of the study was to observe the potential diversity of the oral microbiota among individual patients, sample locations, HIV status and various dental characteristics. We found that there were significant differences in the microbiome among the enrolled patients, and between sampling locations. The analysis was complicated by uneven enrollment in the patient cohorts, with only five HIV-negative patients enrolled in the study and by the rapid improvement in the health of HIV-infected children between the time the study was conceived and completed. The generally good oral health of the HIV-negative patients limited the number of dental plaque samples that could be collected. We did not identify significant differences between well-controlled HIV-positive patients and HIV-negative controls, suggesting that well-controlled HIV-positive patients essentially harbor similar oral flora compared to patients without HIV. Nor were significant differences in the oral microbiota identified between different teeth or with different dental characteristics. Additional studies are needed to better characterize the oral microbiome in children and those with poorly-controlled HIV infections.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G 2 , which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G 2 /M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.

The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P< 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them werecdc42andlyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.

Objective. To characterize the type and frequency of biochemical lipid abnormalities and physical changes in body composition associated with the use of protease inhibitor (PI)-containing antiretroviral therapy among human immunodeficiency virus-infected children treated for up to 6 years. Methods. A retrospective study of human immunodeficiency virus-infected pediatric patients enrolled in research protocols between August 1995 and December 2001 was performed. All patients who had received a PI for ≥2 years as part of their investigational antiretroviral treatment regimens during the study period were eligible. Of the 110 patients identified as having received PI therapy, 94 met the study criteria. Results. Of the 94 patients evaluated, 9 patients (10%) developed fat redistribution as well as dyslipidemia, 49 patients (52%) developed dyslipidemia without associated physical changes, and 36 patients (38%) exhibited no elevation of lipid levels or physical signs of fat redistribution. For all 9 patients with fat redistribution, the onset of the physical changes was closely associated with changes during pubertal development. Fat redistribution was also associated with lower viral loads and higher, more sustained levels of dyslipidemia. The onset of dyslipidemia and fat redistribution peaked between 10 and 15 years of age. Conclusion. Among pediatric patients receiving PI therapy, there seems to be an age range in which children are at greater risk of developing hypercholesterolemia and subsequent fat redistribution, suggesting that unidentified physiologic changes associated with puberty may predispose pediatric patients treated with PI therapy to developing lipodystrophy.

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. In order to study the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This system uses Flp-mediated efficient recombination and tetracycline-inducible expression. The Rta transcriptional activator, which acts as a molecular switch for lytic reactivation of KSHV, was efficiently integrated downstream of the Flp recombination target site, and its expression was tightly controlled by tetracycline. Like stimulation with tetradecanoyl phorbol acetate (TPA), the ectopic expression of Rta efficiently induced a complete cycle of viral replication, including a well-ordered program of KSHV gene expression and production of infectious viral progeny. A striking feature of Rta-mediated lytic gene expression was that Rta induced KSHV gene expression in a more powerful and efficient manner than TPA stimulation, indicating that Rta plays a central, leading role in KSHV lytic gene expression. Thus, our streamlined gene expression system provides a novel means not only to study the effects of viral gene products on overall KSHV gene expression and replication, but also to understand the natural viral reactivation process.

2 Akt (PKB), a pivotal protein kinase which can block apoptosis and promotes cell survival, was identified to be chiefly down-regulated in a dose-dependent manner following CEES treatment. Functional analysis showed that the attendant Akt activity was simultaneously reduced. 3 PDK1, an upstream eAector of Akt, was also down-regulated following CEES exposure, but two other upstream eAectors of Akt, PI3-K and PDK2, remained unchanged. 4 The phosphorylation of Akt at Ser 473 and Thr 308 was significantly decreased following CEES treatment, reflecting the suppressed kinase activity of both PDK1 and PDK2. 5 Concurrently, the anti-apoptotic genes, Bcl family, were down-regulated, in sharp contrast to the striking up-regulation of some death executioner genes, caspase 3, 6, and 8. 6 Based on these findings, a model of CEES-induced apoptosis was established. These results suggest that CEES attacked the Akt pathway, directly or indirectly, by inhibiting Akt transcription, translation, and post-translation modification. 7 Taken together, upon exposure to CEES, apoptosis was induced in Jurkat cells via the downregulation of the survival factors that normally prevent the activation of the death executioner genes, the caspases. British Journal of Pharmacology (2002) 137, 245‐252. doi:10.1038/sj.bjp.0704856

Two children (ages 12 and 13 years) with transfusion-acquired human immunodeficiency virus (HIV) infection presented with facial pain and rhinorrhea. Radiographic imaging showed extensive paranasal sinus disease, presumed to be bacterial sinusitis, and the patients were treated with broad-spectrum oral antibiotics. Both patients were unresponsive to oral agents and were switched to intravenous antibiotics. Despite aggressive antimicrobial therapy, one patient (case 1) developed increased periorbital swelling and proptosis, and the other patient (case 2) developed symptoms of nasopharyngeal obstruction. Repeat imaging showed progression of the infiltrative process extending from the paranasal sinuses into the orbit (case 1), and nasopharynx (case 2). Surgical exploration and tissue biopsies were performed on both patients and the histopathology was consistent with Burkitt's/Burkitt's-like lymphoma. Combination systemic and intrathecal chemotherapy resulted in a complete remission in both patients. These reports illustrate the fact that Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses may initially masquerade as an acute bacterial sinusitis. The ability of the tumor to extend rapidly from the sinuses into the orbit and nasopharynx reinforces the importance of early diagnosis and treatment. Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses has not been previously described in HIV-infected children.

Human herpesvirus 8 (HHV-8), a gammaherpesvirus implicated in Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, encodes several pathogenically important cellular homologs. To define the HHV-8 transcription program, RNA obtained from latently infected body cavity-based lymphoma 1 cells induced to undergo lytic replication was used to query a custom HHV-8 DNA microarray containing nearly every known viral open reading frame. The patterns of viral gene expression offer insights into the replication and pathogenic strategies of HHV-8.

Little is known about cellular immunity to human herpesvirus 8 (HHV-8), the virus associated with Kaposi's sarcoma (KS). T cell proliferative responses to purified HHV-8 were measured in homosexual men, a group with elevated HHV-8 seroprevalence and high risk of KS. None of 20 blood donor controls had T cell responses to HHV-8. Among human immunodeficiency virus (HIV)-negative homosexual men, 8 (42%) of 19 HHV-8 seropositive men responded as did 4 (16%) of 25 HHV-8 seronegative men. Among HIV-positive homosexual men, however, none of 21 HHV-8 seropositives had T cell responses to HHV-8, even though most responded to common recall antigens, and 10 had >/=400 CD4 cells/mm3. The results suggest that HHV-8 T cell proliferative responses are common in HIV-negative homosexual men and that HIV infection may be associated with diminished HHV-8 cellular immunity, possibly before there is substantial depletion of CD4 cells. If correct, this could explain why KS occurs relatively early in HIV infection/AIDS.

Telomere shortening may reflect the total number of divisions experienced by a somatic cell and is associated with replicative senescence. We found that the average rate of telomere shortening in peripheral blood mononuclear cells (PBMCs) obtained longitudinally from nine different infants during the first 3 years of life (270 bp per year) is more than fourfold higher than in adults and does not correlate with telomerase activity. These results show that the rate of telomere loss changes during ontogeny, suggesting the existence of periods of accelerated cell division. Because human immunodeficiency virus (HIV) preferentially infects actively dividing cells, our observation suggesting accelerated cell division in children may provide an explanation for some of the distinctive pathogenic features of the HIV disease in infants, including higher viral loads and more rapid progression to acquired immunodeficiency syndrome (AIDS).

Glycocalicin (GC) is the carbohydrate-rich portion of platelet membrane glycoprotein Ib(alpha) that can be cleaved from circulating platelets by proteases. The plasma GC level is an indicator of platelet turnover. Using an ELISA for GC, we assayed the plasma of 20 normal children (age 6 to 13 years), 50 HIV+ children (ages 4 to 18 years), 32 normal adults (ages 21 to 53 years), and 50 HIV+ adults (ages 24 to 66 years). The results were adjusted for individual platelet counts to give GC indexes (GCI). The normal children and the normal adults had significantly different GCI distributions (P = .002). In both normal and HIV+ individuals the GCI decreased with increasing platelet count (-.73r-.34). Twenty-eight percent of the HIV+ children and 28% of the HIV+ adults had elevated GCI values. The majority of these elevated values occurred in patients with platelet counts100,000/microL. Neither the GCI nor the platelet count was correlated with viral load. The platelet count, however, was weakly correlated with the CD4 count in both children (r = .31) and adults (r = .30) infected with HIV. Also, the CD4 count was weakly and inversely correlated with GCI in HIV+ adults (r = -.34) and in children (r = -.24). We conclude that increased GCI and, by implication, increased platelet turnover is a relatively common feature of advanced HIV disease. Furthermore, GCI may be elevated in HIV+ patients even with a platelet count100,000/microL, suggesting increased platelet turnover before thrombocytopenia develops.

Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. Microglia and macrophages are major HIV-1 targets in the brain, whereas HIV-1 infected neurons or glial cells have been rarely reported. This suggests that indirect mechanisms may account for the severe neuronal damage observed in these patients. Nevertheless, immature, mitotically active neuronal and glial cells, which are present during fetal development, are susceptible to HIV-1 infection and replication in vitro, suggesting that HIV-1 infection during organ development may present unique features. To better characterize virus-host cells interactions in the developing CNS, we have examined the susceptibility of embryologically and biochemically distinct neuronal cell lines to HIV-1 infection. Here we show that mitotically active, immature neurons of distinct lineages, have different susceptibilities to HIV-1 infection and replication and different abilities to support viral gene expression. Mutational analysis of HIV-1 LTR reveals that a region of the viral promoter between nucleotide -255 to -166 is responsible for most quantitative and qualitative differences in viral transactivation among different neuroblasts. This suggests that specific regions of the viral promoter and cellular factors, either lineage- or differentiation-dependent, which bind to those regions, may contribute to control the levels of virus replication and possibly restrict the viral tropism in the developing brain. This may contribute to the establishment of a virus reservoir in the immature CNS and participate by either direct or indirect mechanisms to the severity of the AIDS-related pediatric neurological dysfunction.

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

Latently infected cell reservoirs represent the main barrier to HIV eradication. Combination antiretroviral therapy (cART) effectively blocks viral replication but cannot purge latent provirus. One approach to HIV eradication could include cART to block new infections plus an agent to activate latent provirus. NF-κB activation induces HIV expression, ending latency. Before activation, IκB proteins sequester NF-κB dimers in the cytoplasm. Three canonical IκBs, IκBα, IκBβ, and IκBε, exist, but the IκB proteins' role in HIV activation regulation is not fully understood. We studied the effects on HIV activation of targeting IκBs by single and pairwise small interfering RNA (siRNA) knockdown. After determining the relative abundance of the IκBs, the relative abundance of NF-κB subunits held by the IκBs, and the kinetics of IκB degradation and resynthesis following knockdown, we studied HIV activation by IκB knockdown, in comparison with those of known HIV activators, tumor necrosis factor alpha (TNF-α), tetradecanoyl phorbol acetate (TPA), and trichostatin A (TSA), in U1 monocytic and J-Lat 10.6 lymphocytic latently infected cells. We found that IκBα knockdown activated HIV in both U1 and J-Lat 10.6 cells, IκBβ knockdown did not activate HIV, and, surprisingly, IκBε knockdown produced the most HIV activation, comparable to TSA activation. Our data show that HIV reactivation can be triggered by targeting two different IκB proteins and that IκBε may be an effective target for HIV latency reactivation in T-cell and macrophage lineages. IκBε knockdown may offer attractive therapeutic advantages for HIV activation because it is not essential for mammalian growth and development and because new siRNA delivery strategies may target siRNAs to HIV latently infected cells.

Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based “universal” placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates., IMPORTANCE Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint.

The knowledge of HIV molecular biology has increased rapidly during the past 10 years. This article presents an outline of HIV molecular biology and discusses the stages of the viral life cycle and the many viral and cellular functions required for viral replication. These essential functions serve as targets for existing and potential therapeutic agents; the details of the viral life cycle provide insights into HIV pathogenesis.

To investigate potential mechanisms for HIV-1 proviral latency, we generated a set of chronically HIV-1 infected and stably long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT)-transfected TE671/RD cells, and studied both their virus production and LTR-driven reporter gene expression. Established tissue culture models of retroviral latency in lymphoid and monocytoid cell lines have demonstrated that the induction of virus production is associated with a shift in HIV-1-specific mRNA from a predominance of singly and multiply spliced mRNA's to the production of full-length HIV-1 RNA. We found a similar pattern in TE671/RD cells, but in contrast to U1 and ACH2 cells, could not induce viral replication by exposure to phorbol myristate acetate (PMA) alone. We demonstrated instead that production of full-length viral RNA, viral replication, and LTR-driven CAT expression could be induced by exposure to sodium butyrate. The most proximate effect of sodium butyrate is inhibition of cellular histone deacetylase(s) which results in disruption of nucleosomes relieving one level of restriction to gene expression. Consistent with this mechanism of action, we further found that sodium butyrate's effects: (i) act synergistically with PMA and TNF-alpha; (ii) are independent of protein synthesis; (iii) do not affect the constitutively expressed creatine phosphokinase gene; (iv) do not map to a discrete sequence motif in the viral LTR; and (v) are not blocked by N-acetyl cysteine but (vi) are blocked by novobiocin, an inhibitor of cellular topoisomerase II. These data show that a similar pattern of restricted viral RNA expression exists in this nonlymphoid cellular model of HIV-1 latency. In contrast however, these results suggest that in these cells there is an additional block to viral gene expression, which is overcome with sodium butyrate. These results are discussed in the context of histone-mediated repression of HIV-1 gene expression.

In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S. L. Zeichner, J. Y. H. Kim, and J. C. Alwine, J. Virol. 65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity.

Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21(WAF1) (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21(WAF1) gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.

Several polyadenylylation (PA) signals containing elements upstream of the AAUAAA have recently been characterized. Similar to PA elements found downstream of the AAUAAA, the upstream elements function to increase efficiency of AAUAAA use as a signal for cleavage and PA. Using deletion and linker scanning mutations we show that the PA signal of human immunodeficiency virus type 1 contains upstream elements transcribed from the U3 region of the 3' long terminal repeat. The element that has the greatest effect on PA site use lies 77 to 94 nucleotides upstream of the AAUAAA, between the TATA element and the transcriptional initiation site. Mutations in the adjacent region, between 59 and 76 nucleotides upstream of the AAUAAA, have a smaller effect on PA efficiency. Mutations in a region further upstream, between 141 and 176 nucleotides upstream of the AAUAAA, also affected PA modestly. Functional similarity between upstream elements was indicated by the ability of the human immunodeficiency virus upstream region to replace the upstream region of the simian virus 40 late PA signal. The sequence of the major upstream element of human immunodeficiency virus is uracil-rich, analogous to many defined downstream PA elements. This fact may imply that upstream and downstream elements have similar mechanisms of action.

5 of 6 children infected with human immunodeficiency virus (HIV) receiving Tenofovir disoproxil fumarate (TDF) experienced absolute decreases in bone mineral density (BMD). 2 pre-pubertal subjects experienced >6% BMD decreases. 1 subject was the smallest child and experienced a 27% decrease, necessitating withdrawal of TDF. Subsequently, her BMD recovered. Monitoring of children infected with HIV who require treatment with TDF is warranted.