An approach to the management of unintentional weight loss in elderly people

UNINTENTIONAL WEIGHT LOSS, or the involuntary decline in total body weight over time, is common among elderly people who live at home. Weight loss in elderly people can have a deleterious effect on the ability to function and on quality of life and is associated with an increase in mortality over a 12-month period. A variety of physical, psychological and social conditions, along with age-related changes, can lead to weight loss, but there may be no identifiable cause in up to one-quarter of patients. We review the incidence and prevalence of weight loss in elderly patients, its impact on morbidity and mortality, the common causes of unintentional weight loss and a clinical approach to diagnosis. Screening tools to detect malnutrition are highlighted, and nonpharmacologic and pharmacologic strategies to minimize or reverse weight loss in older adults are discussed.Unintentional weight loss is the involuntary decline in total body weight over time. In clinical practice, it is encountered in up to 8% of all adult outpatients¹ and 27% of frail people 65 years and older.² Weight loss is an important risk factor in elderly patients. It is associated with increased mortality, which can range from 9% to as high as 38% within 1 to 2.5 years after weight loss has occurred.^1,3,4 Frail elderly people,⁵ people with low baseline body weight,^5,6,7 and elderly patients recently admitted to hospital are particularly susceptible to increased mortality.^8,9 Weight loss is also associated with an increased risk of in-hospital complications,^10,11 a decline in activities of daily living or physical function,^12,13 higher rates of admission to an institution^2,8 and poorer quality of life.¹⁴     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Magnetospirillum bellicus sp. nov., a Novel Dissimilatory Perchlorate-Reducing Alphaproteobacterium Isolated from a Bioelectrical Reactor

Previously isolated dissimilatory perchlorate-reducing bacteria (DPRB) have been primarily affiliated with the Betaproteobacteria. Enrichments from the cathodic chamber of a bioelectrical reactor (BER) inoculated from creek water in Berkeley, CA, yielded a novel organism most closely related to a previously described strain, WD (99% 16S rRNA gene identity). Strain VDY^T has 96% 16S rRNA gene identity to both Magnetospirillum gryphiswaldense and Magnetospirillum magnetotacticum, and along with strain WD, distinguishes a clade of perchlorate-reducing Magnetospirillum species in the Alphaproteobacteria. In spite of the phylogenetic location of VDY^T, attempted PCR for the key magnetosome formation genes mamI and mamL was negative. Strain VDY^T was motile, non-spore forming, and, in addition to perchlorate, could use oxygen, chlorate, nitrate, nitrite, and nitrous oxide as alternative electron acceptors with acetate as the electron donor. Transient chlorate accumulation occurred during respiration of perchlorate. The organism made use of fermentation end products, such as acetate and ethanol, as carbon sources and electron donors for heterotrophic growth, and in addition, strain VDY^T could grow chemolithotrophically with hydrogen serving as the electron donor. VDY^T contains a copy of the RuBisCo cbbM gene, which was expressed under autotrophic but not heterotrophic conditions. DNA-DNA hybridization with strain WD confirmed VDY^T as a separate species (46.2% identity), and the name Magnetospirillum bellicus sp. nov. (DSM 21662, ATCC BAA-1730) is proposed.Dissimilatory perchlorate-reducing bacteria (DPRB) use perchlorate as a terminal electron acceptor during respiration, reducing it completely to chloride. As a consequence, bioremediation of perchlorate has been identified as the most effective means of treating this harmful contaminant (10), which, due to historically unregulated release into the environment, has become widespread (13, 20, 41). Fortunately, DPRB are ubiquitous and can be readily isolated from a variety of environments (1, 10, 11, 39, 44), and a key gene in the pathway, the chlorite dismutase (cld) gene, has been broadly detected (6). Much has been revealed about the biochemistry and genetics of microbial perchlorate reduction through the study of several model organisms, including Dechloromonas aromatica and Dechloromonas agitata, by a variety of groups (5, 6, 8, 9, 17, 28, 29, 34, 35, 38, 47, 51, 56, 57).Less is known about the variation in physiology between these organisms or the evolution of the perchlorate reduction metabolism, highlighting a need for further isolation and characterization of pure cultures. The lack of congruence between phylogenetic trees of cld and the 16S rRNA gene among tested DPRB suggests that the metabolism may be the result of horizontal gene transfer (6). Given that various elements of the pathway may be mobile, it is not unreasonable to expect that organisms with a wide phylogenetic diversity could acquire the ability to reduce perchlorate. As more varied enrichment conditions are tested (2, 39), sometimes as a result of novel bioreactor development for perchlorate treatment (38, 40, 45), the true phylogenetic diversity of DPRB is becoming apparent, supporting the hypothesis that the metabolism may be widespread within the tree of life, similar to other respiratory processes, such as the reduction of sulfate, Fe(III), and nitrate.Although perchlorate has been primarily regarded as an anthropogenic contaminant, a variety of studies have indicated that perchlorate occurs naturally (29-31, 34), which provides a possible explanation for the selective pressure behind the evolution of perchlorate reduction genes. As more is understood about the chlorine redox cycle on earth, knowledge about the diversity of organisms capable of interacting with the various oxyanions of chlorine is becoming more important. Here, we report the characterization of a unique DPRB in the Alphaproteobacteria. Strain VDY^T was isolated from the surface of a working electrode in an active perchlorate-reducing bioelectrical reactor (BER) that was inoculated with water from Strawberry Creek on the University of California, Berkeley, campus (40). This is only the second described DPRB in the Alphaproteobacteria, the other being the closely related strain WD (26), and these strains compose a unique clade of perchlorate-reducing organisms in the genus Magnetospirillum.     

Apolipoprotein E ε4 genotype as a risk factor for cognitive decline and dementia: data from the Canadian Study of Health and Aging

Background

Apolipoprotein E (ApoE) ε4 genotype is a well-established risk factor for Alzheimer''s disease (AD). However, its effect on predicting conversion from normal to “cognitive impairment, no dementia” (CIND) and from CIND to AD is less clear.

Methods

We used a nested case–control design from the population-based Canadian Study of Health and Aging (CSHA) to examine the effect of ApoE ε4 genotype on the conversion of subjects from normal to CIND and from CIND to AD. We also contrasted these findings with incident cases of AD and vascular dementia (VaD) in the CSHA cohort.

Results

The ApoE ε4 genotype was a significant risk factor for conversion from CIND to AD and from normal to AD and VaD. However, it was not a significant risk factor for conversion from normal to CIND. This effect is robust to adjustment for age, sex and education level. There is significant interaction between the ApoE ε4 genotype and age for AD and for conversion from CIND to AD. No interaction between ApoE ε4 genotype, sex, age, ethnicity and education level was found in other subgroup analyses. The positive predictive value of ApoE ε4 for predicting CIND conversion to AD was 0.48, and the negative predictive value was 0.65.

Interpretation

Possession of an ApoE ε4 allele increases the risk of AD developing from CIND. It is also associated with a decrease in the age at onset of AD. Its predictive values do not support its utility as a diagnostic test for predicting progression from CIND to AD, but it may be useful in research studies to enrich study samples that have a higher rate of progression to AD.Dementia has a profound impact on patients, families, caregivers and society in general. Data from the Canadian Study of Health and Aging (CSHA) show that 252 600 people had dementia in Canada in 1991; probable Alzheimer''s disease (AD) was diagnosed in 64% of those people.¹ It was also estimated that the net annual cost to society of care for dementia in Canada in 1991 was over $3.9 billion.² The prevalence of AD rises exponentially, doubling approximately every 5 years between the ages of 65 and 85. In recent years, rapid progress in molecular genetics has fostered the discovery of at least 4 genes associated with AD: the amyloid precursor protein (APP), the presenilin-1 gene (PS-1), the presenilin-2 gene (PS-2) and the apolipoprotein E gene (ApoE).^3,4,5Mutations in APP, PS-1 and PS-2 account for virtually all autosomal dominant inherited early-onset forms of AD. However, this form of AD represents less than 10% of all AD cases. By contrast, ApoE ε4 polymorphism does not cause AD, but it operates as a susceptibility gene or genetic risk factor. The gene exists in 3 different allele polymorphisms — ε2, ε3 and ε4 — in the general population. From previous epidemiological studies, it is estimated that people who carry 1 ε4 allele are 3 times more likely to have AD than those who do not carry any ε4 allele, and those who carry 2 ε4 alleles are 9 times more likely to develop AD than those who do not.^6,7 In addition, the ε4 allele appears to exert maximal effect in patients in whom AD is diagnosed between the ages of 55 and 75.^8,9 The ApoE ε4 allele also has been implicated as a risk factor for vascular dementia (VaD), but the findings have been inconsistent, with some studies showing positive association^{10,11,12,13,14} and others not.^{15,16,17,18,19} Recently, it has been recognized that patients who have “cognitive impairment but no dementia” (CIND) are an important group at risk for dementia. Few studies have examined ApoE ε4 as a predictor for progression from normal to CIND and from CIND to dementia.^17,19,20 To further define the relation between ApoE ε4 polymorphism and the risk of dementia in the Canadian population, we examined ApoE ε4 genotype as a predictor for conversion from normal to CIND and from CIND to AD or VaD using data obtained from the CSHA cohort. We also investigated the role of ApoE ε4 genotype as a risk factor for incident cases of AD and VaD, while controlling for the effects of age, sex and level of education.     

Inhibition of Selenium Metabolism in the Oral Pathogen Treponema denticola

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.The biological use of selenium as a catalyst, incorporated into proteins as selenocysteine, is broad. It plays an essential role in energy metabolism, redox balance, and reproduction in a variety of organisms, from bacterial pathogens to eukaryotic parasites to humans. The results of several epidemiological studies indicate that higher levels of selenium in the mammalian diet can have a negative effect on dental health (2, 17-19, 39). Although the impact of selenium is attributed to its influence on the physical properties of the enamel surface (10), the role of selenium in supporting the oral microbial community has not been studied.The oral cavity is a highly complex microbiome, with a large proportion of its residents uncharacterized due to their fastidious nature and resistance to traditional culture methods (11). Analysis of whole saliva indicates that bacterial metabolism influences the amino acid composition and indicates a role for amino acid fermentation (38). Curtis et al. demonstrated the occurrence of Stickland reactions in dental plaque (9). These reactions were first described in clostridia (35-37). They involve the coupled fermentation of amino acids in which one amino acid is oxidized (Stickland donor) and another (Stickland acceptor) is reduced (29). Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1, 7, 8, 15, 16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin. For the organisms that use this complex, this is a vital source of ATP. Thus far, the requirement for selenocysteine at the active site of this enzyme complex is universally conserved, even though all other selenoproteins that have been identified using computational techniques have a putative cysteine homologue (24).Treponema denticola is considered one of the primary pathogens responsible for periodontitis, a chronic inflammatory disease that is the major cause of adult tooth loss (11, 27, 33). It is the best-studied oral spirochete, commonly found with other spirochetes within the periodontal pocket. It expresses a variety of virulence factors and is capable of adhering to and penetrating endothelial cell monolayers (31). Its health impact may reach beyond the oral cavity. A recent study linked periodontitis with peripheral arterial disease and detected T. denticola, along with other periodontal pathogens, in atherosclerotic plaque (3). Sequence analysis indicates the presence of several selenoproteins in addition to glycine reductase within the genome of T. denticola (24). This organism exhibits a strict growth requirement for selenium (32).A significant literature exists that clearly demonstrates the antimicrobial activity of fluoride compounds against microorganisms associated with dental decay and periodontitis. Both sodium fluoride and stannous fluoride, as well as stannous ions alone, inhibit the growth of T. denticola (21). The inhibitory effect of stannous salts on T. denticola''s growth is unexplained. It should be noted that toothpastes containing stannous fluoride are more effective in reducing gingivitis and plaque (28, 30).Tin, as well as several other trace elements, modulates the effects of acute selenium toxicity (20). Conversely, selenium affects the activity of tin in animal models (4-6). In this study, we examine the possibility that stannous ions interfere with selenium metabolism in T. denticola.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate by Granular Biofilms Comprising a Mixture of Microbes

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day⁻¹ g⁻¹, with reduction of 0.17 mM day⁻¹ g⁻¹ under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.Chromium is a common industrial chemical used in tanning leather, plating chrome, and manufacturing steel. The two stable environmental forms are hexavalent chromium [Cr(VI)] and trivalent chromium [Cr(III)] (20). The former is highly soluble and toxic to microorganisms, plants, and animals, entailing mutagenic and carcinogenic effects (6, 22, 33), while the latter is considered to be less soluble and less toxic. Therefore, the reduction of Cr(VI) to Cr(III) constitutes a potential detoxification process that might be achieved chemically or biologically. Microbial reduction of Cr(VI) seemingly is ubiquitous; Cr(VI)-reducing bacteria have been isolated from both Cr(VI)-contaminated and -uncontaminated environments (6, 7, 23, 38, 39). Many archaeal/eubacterial genera, common to different environments, reduce a wide range of metals, including Cr(VI) (6, 16, 21). Some bacterial enzymes generate Cr(V) by mediating one-electron transfer to Cr(VI) (1, 4), while many other chromate reductases convert Cr(VI) to Cr(III) in a single step.Biological treatment of Cr(VI)-contaminated wastewater may be difficult because the metal''s toxicity potentially can kill the bacteria. Accordingly, to protect the cells, cell immobilization techniques were employed (31). Cells in a biofilm exhibit enhanced resistance and tolerance to toxic metals compared with free-living ones (15). Therefore, biofilm-based reduction of Cr(VI) and its subsequent immobilization might be a satisfactory method of bioremediation because (i) the biofilm-bound cells can tolerate higher concentrations of Cr(VI) than planktonic cells, and (ii) they allow easy separation of the treated liquid from the biomass. Ferris et al. (11) described microbial biofilms as natural metal-immobilizing matrices in aqueous environments. Bioflocs, the active biomass of activated sludge-process systems are transformed into dense granular biofilms in sequencing batch reactors (SBRs). As granular biofilms settle extremely well, the treated effluent is separated quickly from the granular biomass by sedimentation (9, 24). Previous work demonstrated that aerobic granular biofilms possess tremendous ability for biosorption, removing zinc, copper, nickel, cadmium, and uranium (19, 26, 31, 32, 40). However, no study has investigated the role of cellular metabolism of aerobically grown granular biofilms in metal removal experiments. Despite vast knowledge about biotransformation by pure cultures, very little is known about reduction and immobilization by mixed bacterial consortia (8, 12, 13, 16, 20, 31, 36). Our research explored, for the first time, the metabolically driven removal of Cr(VI) by microbial granules.The main aim of this study was to investigate Cr(VI) reduction and immobilization by mixed bacterial consortia, viz., aerobically grown granular biofilms. Such biofilm-based systems are promising for developing compact bioreactors for the rapid biodegradation of environmental contaminants (17, 24, 29). Accordingly, we investigated the microbial reduction of Cr(VI) by aerobically grown biofilms in batch and fed-batch experiments and analyzed the oxidation state and association of the chromium immobilized on the biofilms by X-ray absorption near edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS).     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.