Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition.

The inflammasome is hypothesized to be a key mediator of the response to physiological and psychological stressors, and its dysregulation may be implicated in major depressive disorder. Inflammasome activation causes the maturation of caspase-1 and activation of interleukin (IL)-1ß and IL-18, two proinflammatory cytokines involved in neuroimmunomodulation, neuroinflammation and neurodegeneration. In this study, C57BL/6 mice with genetic deficiency or pharmacological inhibition of caspase-1 were screened for anxiety- and depressive-like behaviors, and locomotion at baseline and after chronic stress. We found that genetic deficiency of caspase-1 decreased depressive- and anxiety-like behaviors, and conversely increased locomotor activity and skills. Caspase-1 deficiency also prevented the exacerbation of depressive-like behaviors following chronic stress. Furthermore, pharmacological caspase-1 antagonism with minocycline ameliorated stress-induced depressive-like behavior in wild-type mice. Interestingly, chronic stress or pharmacological inhibition of caspase-1 per se altered the fecal microbiome in a very similar manner. When stressed mice were treated with minocycline, the observed gut microbiota changes included increase in relative abundance of Akkermansia spp. and Blautia spp., which are compatible with beneficial effects of attenuated inflammation and rebalance of gut microbiota, respectively, and the increment in Lachnospiracea abundance was consistent with microbiota changes of caspase-1 deficiency. Our results suggest that the protective effect of caspase-1 inhibition involves the modulation of the relationship between stress and gut microbiota composition, and establishes the basis for a gut microbiota-inflammasome-brain axis, whereby the gut microbiota via inflammasome signaling modulate pathways that will alter brain function, and affect depressive- and anxiety-like behaviors. Our data also suggest that further elucidation of the gut microbiota-inflammasome-brain axis may offer novel therapeutic targets for psychiatric disorders.

CNS-specific regulatory elements in brain-derived HIV-1 strains affect responses to latency-reversing agents with implications for cure strategies.

Latency-reversing agents (LRAs), including histone deacetylase inhibitors (HDACi), are being investigated as a strategy to eliminate latency in HIV-infected patients on suppressive antiretroviral therapy. The effectiveness of LRAs in activating latent infection in HIV strains derived from the central nervous system (CNS) is unknown. Here we show that CNS-derived HIV-1 strains possess polymorphisms within and surrounding the Sp transcription factor motifs in the long terminal repeat (LTR). These polymorphisms result in decreased ability of the transcription factor specificity protein 1 to bind CNS-derived LTRs, reducing the transcriptional activity of CNS-derived viruses. These mutations result in CNS-derived viruses being less responsive to activation by the HDACi panobinostat and romidepsin compared with lymphoid-derived viruses from the same subjects. Our findings suggest that HIV-1 strains residing in the CNS have unique transcriptional regulatory mechanisms, which impact the regulation of latency, the consideration of which is essential for the development of HIV-1 eradication strategies.

Hepatitis C seropositivity is not a risk factor for sensory neuropathy among patients with HIV.

BACKGROUND: Sensory neuropathy (SN) is common in patients with HIV. Hepatitis C (HCV) coinfection is often cited as an HIV-SN risk factor, but data to support this are lacking. This collaboration aimed to examine the association between HCV serostatus and SN risk among ambulatory HIV-positive patients. METHODS: Patients with HIV were assessed in cross-sectional studies in Baltimore, Jakarta, Johannesburg, Kuala Lumpur, Melbourne, and Sydney for SN (defined by both supportive symptoms and signs). HCV seropositivity was assessed as an SN risk using a chi(2) test, followed by logistic regression modeling to correct for treatment exposures and demographics. RESULTS: A total of 837 patients of African, Asian, and Caucasian descent were studied. HCV seroprevalence varied by site (Baltimore n = 104, 61% HCV+; Jakarta 96, 51%; Johannesburg 300, 1%; Kuala Lumpur 97, 10%; Melbourne 206, 16%; Sydney 34, 18%). HCV seropositivity was not associated with increased SN risk at any site, but was associated with reduced SN risk in Melbourne (p = 0.003). On multivariate analyses, the independent associations with SN were increasing age, height, and stavudine exposure. HCV seropositivity was not independently associated with an increased SN risk at any site, but associated independently with reduced SN risk in Baltimore (p = 0.04) and Melbourne (p = 0.06). CONCLUSIONS: Hepatitis C (HCV) seropositivity was not associated with increased sensory neuropathy risk among HIV-positive patients at any site. While we were unable to assess HCV RNA or liver damage, the data suggest that HCV coinfection is not a major contributor to HIV-SN. HCV = hepatitis C; SN = sensory neuropathy.

Prevalence of and risk factors for HIV-associated neuropathy in Melbourne, Australia 1993-2006.

OBJECTIVES: The aim of the study was to describe the prevalence of and risk factors for HIV-associated sensory neuropathy (HIV-SN) in 2006 [the era of stavudine, didanosine and zalcitabine (dNRTI)-sparing highly active antiretroviral therapy (HAART)] and to compare our findings with data obtained in the same clinic in 1993 (pre-HAART) and 2001 (frequent use of dNRTI-containing HAART). METHODS: This was a cross-sectional comparative study using convenience sampling. HIV-positive adults attending a tertiary referral clinic over a 2-week period were screened for HIV-SN using the AIDS Clinical Trials Group screening tool. HIV-SN was defined as present if the patient had both neuropathic symptoms and abnormal signs. Demographic, clinical, laboratory and treatment data were considered as possible risk factors for HIV-SN, and results were compared with data obtained in the same clinic in 1993 and 2001. RESULTS: One hundred patients were screened. The prevalence of HIV-SN was 42%, which was unchanged since 2001 (44%) despite a significant reduction in the use of dNRTIs. HIV-SN remained much more common than in 1993 (42% vs 13%; P<0.0001). The only independent associations with HIV-SN in 2006 were increasing patient age and a history of exposure to either stavudine or indinavir. This compares with 1993 when neuropathy was increased in those with Mycobacterium avium complex infection, and 2001 when patient age and use of stavudine and didanosine were the independent associations with HIV-SN in this clinic. CONCLUSIONS: HIV-SN remained common among ambulatory patients in 2006 (42% prevalence) despite a significant reduction in the use of dNRTIs. In addition to patient age and stavudine exposure, indinavir use may be a risk factor for HIV-SN.

Antiretroviral use and other risks for HIV-associated neuropathies in an international cohort.

OBJECTIVE: To explore the association between specific nucleoside reverse transcriptase inhibitors and sensory neuropathies (SNs) and define the modifying roles of hepatitis C (HCV), vitamin B12 deficiency, and impaired glucose tolerance. METHODS: The authors conducted a prospective cohort study of 147 HIV-infected adults at two sites chosen to emphasize demographic differences. Standardized assessments included detailed antiretroviral histories, neurologic examinations, skin biopsies for epidermal nerve quantitation, and quantitative sensory testing. RESULTS: There were significant differences between subjects at Johns Hopkins University (JHU) and Monash University (MU) in gender, race, HIV transmission route, and HCV seroprevalence. Symptomatic SN was present in 49% at JHU and 55% at MU (chi2 = 4.02, p = 0.134) and was significantly more common in those at least age 40 than younger patients (odds ratio [OR] = 2.87, 95% CI = 1.27, 6.49). After adjusting for site, age, and CD4 cell count, exposure to didanosine (ddI) or stavudine (d4T) was associated with an significantly increased likelihood of symptomatic SN (ddI: OR = 3.21, 95% CI: 1.56, 6.60; d4T: OR = 7.66, 95% CI: 2.89, 20.33). Plasma HIV RNA, lactate, and HCV were not associated with SN. Quantitative vibratory testing identified neuropathy with a positive predictive value of 76% and epidermal nerve fiber densities 59%. CONCLUSIONS: Exposure to stavudine and didanosine was significantly associated with a heightened risk for symptomatic sensory neuropathy. Reduced vibration thresholds and epidermal nerve fiber densities had the highest diagnostic efficiency of the laboratory indicators of neuropathy examined, but were relatively insensitive in isolation.

Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination.

Plant-made oral vaccines have the potential to overcome many of the limitations of traditional vaccines. Here we report on progress towards a lettuce-made measles vaccine. Lettuce is a palatable species which exhibits rapid growth in contained hydroponic systems and produces negligible quantities of toxins. Measles virus hemagglutinin (MV-H) protein was successfully expressed in transgenic lettuce and found to be immunogenic in mice. Lettuce extracts containing MV-H protein induced MV neutralising antibodies following intraperitoneal injection and intranasal inoculation of mice. Using a sequential prime-boost strategy in which mice were vaccinated with MV-H DNA followed by an orally delivered freeze-dried MV-H lettuce formulation a 10-fold increased in MV-specific IgG titers was observed relative to mice vaccinated with control lettuce formulations (p=0.05). MV-H protein was stable in freeze-dried lettuce for up to 13 months at room temperature, and survived at least a week at temperatures as high as 50 degrees C. This research represents a significant step towards the development of measles vaccine formulation that is effective, temperature-stable, easy to administer in a resource-poor setting and amenable to large scale manufacture.

Crude saponins improve the immune response to an oral plant-made measles vaccine.

Millions of people live in areas where infectious diseases, such as measles, are endemic and resources are scarce. Heat-stable vaccines that are delivered orally will greatly enhance vaccination programs in these areas. A stumbling block in the development of oral vaccines is the availability of safe and effective mucosal adjuvants, especially for use with subunit vaccines. The experiments presented here examine the ability of CTB/CT, LT(R192G) and crude Quillaja saponin extracts to stimulate MV-specific immune responses in mice, following oral immunisation with plant-made measles virus hemagglutinin (MV-H) protein. LT(R192G) and crude saponin extracts both functioned as potent mucosal adjuvants when ad-mixed with plant-made MV-H protein, and were more effective than CTB/CT. MV-H protein supplemented with saponin extract induced the strongest MV-specific responses, in the greatest number of mice. Crude saponins are routinely used by the food and beverage industry at concentrations greater than those required for adjuvanticity, and as such, they have a better safety profile than bacterial enterotoxins. This study demonstrates their potential as adjuvants for use with oral plant-made vaccines.

The development of a plant-based vaccine for measles.

Plant-based vaccination strategies have the potential to overcome the limitations of the current measles vaccine. The measles virus hemagglutinin (MV-H) protein has been expressed in tobacco. Oral immunisation of mice with plant-derived MV-H protein resulted in MV-specific antibodies and secretory IgA, indicative of humoral and mucosal immune responses. In addition, boosting with oral plant-derived MV-H protein following a MV-H DNA prime, resulted in a greater response than could be induced with either vaccine alone. Collectively, this research represents a significant step towards an effective oral measles vaccine that would be temperature-stable, easy to administer and amenable to inexpensive manufacture.

Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction.

BACKGROUND: With decreased rates of HIV mortality and disease progression attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity. OBJECTIVES: The objectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish the utility of subcutaneous fat as a source of mtDNA for quantification. STUDY DESIGN: PBMC were isolated from blood by centrifugation over Ficoll-Paque and subcutaneous fat was obtained from two 3 mm punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR). RESULTS: The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409+/-148 and 2042+/-391 copies of mtDNA per cell, respectively. CONCLUSIONS: From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat.

Does plasma HIV RNA predict outcome in a cohort of treated HIV-infected individuals followed over 3 years?

BACKGROUND: Despite reductions in AIDS illness and mortality, it is increasingly apparent that a significant proportion of individuals treated with combination antiretroviral (cARV) therapy have continuing or recrudescent HIV RNA in plasma. The predictive value of plasma HIV RNA in treated individual remains uncertain and rates of and risk factors for adverse outcomes such as hospitalisation, opportunistic infections and deaths are needed. OBJECTIVES: The objectives of this study were to establish a retrospective cohort of individuals treated with cARVs, to assess factors associated with detectable HIV RNA and to determine rates of and risk factors for hospitalisation, opportunistic infection and mortality over 3 years of follow-up. STUDY DESIGN: All individuals treated at The Alfred Hospital, Melbourne, Victoria between January and June 1997 who had had plasma HIV RNA measured were included in the retrospective cohort. Clinical, virological and hospitalisation data were recorded and validated by cross-reference with electronically stored laboratory, hospital activity and state notification databases. Outcome was assessed at October 2000. RESULTS: Amongst the 555 individuals tested, 438 (60.7%) had detectable (>500 copies/ml) HIV RNA (bDNA assay, version 2) at baseline. The overall mortality rate was 5.5 per 100 person years; the AIDS rate 1.99 per 100 person years and hospitalisation rate 16.4 per 100 person years. Risk factors for death in this population identified by univariate analysis were HIV RNA concentration at baseline and at follow-up October 2000, nadir and most recent CD4 lymphocyte number, not receiving cARV as initial treatment, total number of ARV agents and number of changes in ARV per year, developing AIDS and being hospitalised during follow-up. In a multivariate model, the most recent CD4 lymphocyte number, the number of different ARVs per year and having more than one hospitalisation remained predictive of death. CONCLUSIONS: HIV RNA remained detectable in the majority (60.7%) of this treatment-experienced population over 3 years, yet mortality rate remained relatively low at 5.5 per 100 person years. Factors associated with death were immunological (CD4 lymphocyte number) and treatment related (numbers of changes of ARV and hospitalisation) rather than virological (HIV RNA) in this cohort. We believe hospitalisation rates may be a useful marker of HIV disease in cARV treated populations and may identify groups at risk of poorer outcome and in need of intervention.

Microglia in HIV-associated neurological diseases.

Human immunodeficiency virus type-1 (HIV-1) is a neurotropic virus linked to a variety of progressive neurologic disorders. This review describes our current understanding of how HIV-1 enters the nervous system and interacts with neuronal and non-neuronal cells to initiate and sustain neurologic dysfunction. The overwhelming majority of cells infected with HIV-1 in the nervous system are microglia/macrophages. Microglial/macrophage infection leads to immune dysregulation as well as production and release of cytotoxic molecules. Interaction of these infected cells with astrocytes may accelerate neurotoxic mechanisms. A hypothetical scenario for how HIV-1 infection leads to neurologic disease is presented.

Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia.

The pathogenesis of HIV-associated dementia (HIVD) has been postulated to be due to the indirect effects of HIV infection, including the aberrant central nervous system production of cytokines and other neurotoxins. A correlation between the severity of dementia and production of neurotoxins in HIVD has been demonstrated. We have previously identified nonproductive HIV infection of astrocytes. Because astrocytes participate in the inactivation of neurotoxins, we hypothesize that HIV nonproductive infection of astrocytes may lead to an environment in which there is a significant level of astrocyte apoptosis and a consequent increase in the levels of neurotoxins and that this results in more rapidly progressing dementia. Postmortem brain tissue was examined from clinically well-characterized HIV-positive demented patients, HIV-positive nondemented patients, and HIV-seronegative nondemented control subjects. The HIVD group was further categorized into subjects with rapid and those with slow progression of dementia. Tissue was paraformaldehyde fixed and paraffin embedded, and 6-microm sections from the basal ganglia and mid-frontal gyrus were processed to detect apoptosis by in situ transferase dUTP nick end labeling. Astrocytes were co-localized using immunohistochemical techniques. In situ polymerase chain reaction (PCR) techniques were utilized to detect HIV DNA in astrocytes. The density of apoptotic astrocytes was significantly greater in the HIV-positive groups than in the HIV-negative group (p < 0.01). The HIVD rapid progressors had a significantly greater number of apoptotic astrocytes in the basal ganglia than did the HIVD slow progressors (p < 0.05). In addition, there were a greater number of HIV DNA-positive astrocytes, as demonstrated by in situ PCR, in the HIVD rapid progressors than in the slow progressor and HIV-nondemented groups. These data suggest that there is an increased rate of astrocyte loss in the subjects with rapidly progressive dementia, in association with an increased number of HIV DNA-positive astrocytes. The results emphasize the importance of understanding more completely the role of HIV infection of astrocytes in the neuropathogenesis of HIVD.

Immunopathogenesis of HIV-associated dementia.

This review provides a subjective analysis of the advances in our understanding of the immunopathogenesis of HIV-associated dementia that have occurred over the past 12 months. The review will focus on the following areas: (i) the role of chemokines and cytokines; (ii) the role of astrocytes, astrocyte cell death and non-productive infection of astrocytes; (iii) a model of the neuropathogenesis of HIV-associated dementia and its impact on treatment paradigms and future research. The requirements for the development of HIV-associated dementia are immunosuppression, the loss of macrophage regulation, central nervous system HIV infection of microglia and macrophages with a neurovirulent HIV strain, restricted HIV infection of astrocytes, and astrocyte cell death, all of which lead to an intracellular milieu that is neurotoxic. This cascade can be prevented and probably reversed by the use of highly active antiretroviral therapy, which controls viral replication both systemically and centrally. However, for those patients who have resistant virus and persistently high levels of replication, or who develop resistance or toxicity, other treatment strategies need to be developed. The control of excessive microglial and macrophage activation or a diminution of astrocyte and neuronal apoptosis could have benefits in terms of cognitive function. We therefore need to develop further our understanding of the immunopathogenesis of HIV-associated dementia so that we can control a number of other steps in the cascade rather than simply controlling the viral replication.

Psychosocial factors are associated with prolonged hospitalization in a population with advanced HIV.

Our aim was to define a subgroup of patients with HIV at risk of adverse outcomes in terms of psychosocial factors in order to improve the targeting of hospital resources. The International Classification of Diseases, 9th Revision (ICD-9) coded discharges of all inpatients with HIV discharged from a tertiary hospital between July 1996 and March 1999 were matched against variables in the HIV/AIDS database. A 'prolonged hospitalization' subgroup was defined as those patients whose cumulative length of stay exceeded 90 days in the 33-month period. There were 2778 non-day stay discharges (n=757 patients) constituting 21,286 bed-days. The prolonged hospitalization group (n=62) accounted for 44.3% of the bed-days. Psychosocial co-diagnoses were associated with prolonged hospitalization in both crude and adjusted logistic analyses. These included psychiatric diagnoses such as mania, psychosis and anxiety, HIV dementia, housing issues and the need for social work interventions. In conclusion, a small group of individuals at risk of adverse outcomes has been defined by markers of psychosocial dysfunction. Increased understanding of this group should enable the development of programmes directed at morbidity and mortality.

Herpes simplex replication and dissemination is not increased by corticosteroid treatment in a rat model of focal Herpes encephalitis.

Neurological damage in Herpes simplex type 1 encephalitis results from neuronal cell death secondary to viral invasion, and from inflammatory changes and cerebral oedema secondary to the immune response to the virus. Corticosteroids could have an important role in the management of Herpes simplex encephalitis because their anti-inflammatory action reduces cerebral oedema. However their use has been limited by concerns that their immunosuppressive actions could increase viral replication and spread. The present study examined this issue in a rat model in which injection of HSV-1 into the cervical vagus nerve produced a well-defined focal encephalitis, characterised by an orderly progression of the virus through central neural pathways connected with vagal afferent termination sites in the medulla oblongata. After injection of HSV-1, rats were treated twice a day, either with vehicle (saline, 400 microl i.p.), with acyclovir (30 mg/kg i.p.), with dexamethasone (5 mg/kg i.p.), or with both acyclovir and dexamethasone. Animals were sacrificed after 72 h, and viral load in different brain regions was quantified by computer-assisted measurement of the area occupied by immunohistochemical reaction product. Treatment with acyclovir reduced viral load to 17 +/- 5% of the saline value (P < 0.01). After dexamethasone treatment, the viral load (63 +/- 13% of the saline value) was also reduced (P < 0.05). Treatment with both acyclovir and dexamethasone reduced viral load to 26 +/- 8% of the saline value (P < 0.01 compared with saline, and P > 0.05 compared to acyclovir alone). Our results confirm the effectiveness of acyclovir in a new model of HSV-1 infection, and provide evidence that corticosteroids do not inhibit the antiviral action of acyclovir. In addition corticosteroids may decrease the extent of infection in their own right. The acute time course studied in our model parallels the time course of acute Herpes simplex encephalitis in humans. Our data suggests that corticosteroids are not detrimental when combined with acyclovir in the management of this condition.