From gut dysbiosis to altered brain function and mental illness: mechanisms and pathways.

The human body hosts an enormous abundance and diversity of microbes, which perform a range of essential and beneficial functions. Our appreciation of the importance of these microbial communities to many aspects of human physiology has grown dramatically in recent years. We know, for example, that animals raised in a germ-free environment exhibit substantially altered immune and metabolic function, while the disruption of commensal microbiota in humans is associated with the development of a growing number of diseases. Evidence is now emerging that, through interactions with the gut-brain axis, the bidirectional communication system between the central nervous system and the gastrointestinal tract, the gut microbiome can also influence neural development, cognition and behaviour, with recent evidence that changes in behaviour alter gut microbiota composition, while modifications of the microbiome can induce depressive-like behaviours. Although an association between enteropathy and certain psychiatric conditions has long been recognized, it now appears that gut microbes represent direct mediators of psychopathology. Here, we examine roles of gut microbiome in shaping brain development and neurological function, and the mechanisms by which it can contribute to mental illness. Further, we discuss how the insight provided by this new and exciting field of research can inform care and provide a basis for the design of novel, microbiota-targeted, therapies.

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.

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.

Prolonged Wait Time Prior to Entry to Home Care Packages Increases the Risk of Mortality and Transition to Permanent Residential Aged Care Services: Findings from the Registry of Older South Australians (ROSA).

BACKGROUND: Older Australians prefer to live in their own homes for longer and reforms have attempted to increase the volume of home care packages (HCPs) accordingly but there remains a queue with the longer-term consequences unclear. OBJECTIVES: This study aims to characterise older Australians according to their wait times for a home care package (HCP), evaluate the association between wait time and mortality and evaluate the association between wait time and transition to permanent residential aged care services after HCP. DESIGN: A retrospective cohort study using data from the National Historical cohort (2003-2014) of the Registry of Older South Australians (ROSA) was conducted. SETTING: Home based aged care services, national cohort. METHODS: Wait time was estimated from approval date to date of receiving a HCP. Descriptive, survival estimates (95% confidence intervals (CIs)), and multivariable survival analyses (Cox-regression) were conducted to evaluate the risk of mortality and transition to permanent residential aged care services by quartiles of wait time for HCP. RESULTS: The cohort was followed for 4.0 years (interquartile range IQR (1.8-7.2)) and 38% were alive at the end of the study period with a median wait time for HCP of 62 (21-187) days. From 178,924 older people who received a HCP during the study period (2003-2013), 33.2% people received HCP within 30 days, 74.3% within 6 months and 25.7% after 6 months. The effect of wait time on risk of mortality was time-dependent, with longer wait times associated with higher mortality in the longer term. Compared to people who waited ≤30 days for a HCP, individuals who waited more than 6 months had an almost 20% excess risk of death (adjusted hazard ratio (aHR), 95%CI = (1.18, 1.16-1.21)) 2 years after entry into a HCP. Those who waited more than 6 months also had a 10% (1.10, 1.06-1.13) higher risk of transition to permanent residential aged care services after two years. CONCLUSION: Prolonged wait times for HCP is associated with a higher risk of long-term mortality as well as transition to permanent residential aged care. It remains to be seen if a shortening of this wait time translates into better health outcomes.

Brain endothelial cell infection in children with acute fatal measles.

Neurologic diseases are important complications of measles. The role of virus infection of the central nervous system as well as the route of virus entry has been unclear. Five autopsied cases of individuals who died with severe acute measles 3-10 d after the onset of the rash were studied for evidence of viral involvement of the central nervous system. In all cases, in situ hybridization and RT-PCR in situ hybridization techniques showed endothelial cell infection. Immunoperoxidase staining with an anti-ferritin antibody revealed a reactive microgliosis. These data suggest that endothelial cells in the brain are frequently infected during acute fatal measles. This site of infection may provide a portal of entry for virus in individuals who subsequently develop subacute sclerosing panencephalitis or measles inclusion body encephalitis and a target for immunologic reactions in post-measles encephalomyelitis.

Projections from rabbit caudal medulla to C1 and A5 sympathetic premotor neurons, demonstrated with phaseolus leucoagglutinin and herpes simplex virus.

We combined Phaseolus vulgaris leucoagglutinin anterograde tracing and Herpes simplex virus transneuronal retrograde tracing to determine whether neurons in the vasodepressor region of the rabbit caudal ventrolateral medulla project to brainstem neurons containing the virus after its transneuronal transport from the adrenal medulla. Five days after adrenal injection of virus, 764 +/- 159 virus-positive neurons were found bilaterally in the brainstem: 61% in the C1 sympathoexcitatory region of the rostral ventrolateral medulla, 30% in the A5 region, 5% in the parapyramidal region, and 3% in the paraventricular nucleus of the hypothalamus. Many of the virus-positive neurons in the C1 and A5 areas also contained tyrosine hydroxylase and, in the parapyramidal area, many contained 5-hydroxytryptamine. After iontophoretic deposit of leucoagglutinin into the vasodepressor region of the caudal ventrolateral medulla, brain regions containing varicose processes labeled with leucoagglutinin included the regions containing virus-positive neurons. We examined the C1 and A5 regions following injections of both tracers in the same rabbits, leucoagglutinin into the caudal ventrolateral medulla and virus into the adrenal gland. Varicosities containing leucoagglutinin were seen in contiguity with perikarya and dendritic branches of neurons containing HSV1, in both the C1 and A5 regions. Studies also revealed labeled varicosities in contiguity with TH-containing C1 and A5 neurons. The projection from the caudal medulla to presumed sympathetic premotor neurons in the C1 area, including some C1 cells, represents a potential pathway whereby activity of neurons in the caudal medulla could reduce blood pressure by inhibiting sympathoexcitatory neurons in the rostral medulla.

Clinical-neuropathologic correlation in HIV-associated dementia.

The structural abnormalities that correlate with the clinical manifestations of HIV-associated dementia (HIVD) are unclear. In a prospectively categorized group of patients with and without HIVD who were followed to autopsy, we correlated HIV-related neuropathologic changes with the presence and severity of HIVD. We also assessed the effect of antiretroviral therapy on the neuropathologic changes. Finally, using reverse transcriptase-polymerase chain reaction on homogenized brain tissue, we correlated the relative expression of mRNA for tumor necrosis factor-alpha (TNF-alpha) with cognitive impairment and with the patterns of neuropathologic changes. The presence of multinucleated giant cells and diffuse myelin pallor were specific for HIVD, but these pathologic changes occurred in only 50% of patients with dementia. Patients treated with antiretroviral agents for > 12 months were less likely to show multinucleated giant cells or diffuse myelin pallor. Levels of mRNA for TNF-alpha from frontal subcortical white matter were significantly greater in patients with HIVD than in AIDS patients without dementia or in seronegative controls. We conclude that routine histopathologic examination of the brain fails to detect multinucleated giant cells and diffuse myelin pallor in 50% of patients dying with HIVD. This suggests that more subtle neuropathologic correlates for the clinical manifestations of HIVD exist. Our observations of elevated levels of TNF-alpha mRNA in HIVD indicate that indirect mechanisms of brain dysfunction, such as abnormal cytokine expression, may contribute to the pathogenesis of HIVD.

Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation.

Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.

Cellular localization of tumor necrosis factor mRNA in neurological tissue from HIV-infected patients by combined reverse transcriptase/polymerase chain reaction in situ hybridization and immunohistochemistry.

HIV-induced neurological disease is postulated to be caused by indirect mechanisms. Tumor necrosis factor (TNF)alpha is increased in the brains in human immunodeficiency virus (HIV)-associated dementia and in the spinal cord in vacuolar myelopathy and may play a pathogenetic role in these diseases. Microglia, astrocytes and infiltrating macrophages can be induced to produce TNF alpha and each has been identified as a source of TNF alpha in neurological disease. Reverse transcriptase synthesis of cDNA and polymerase chain reaction amplification of the cDNA was combined with immunocytochemistry to identify the cellular source of TNF alpha in HIV-induced neurological disease. Cells positive for TNF alpha mRNA were more abundant in white matter than gray matter of the brain from demented individuals. TNF alpha mRNA-positive cells in brains and spinal cords were almost exclusively macrophage-lineage cells. Only rare TNF alpha mRNA-positive cells were astrocytes. We conclude that macrophage-lineage cells are the primary source of elevated central nervous system TNF alpha mRNA in providing further evidence that macrophage activation is an important element in the pathogenesis of HIV-associated neurological disease.

Production and role of cytokines in the CNS of mice with acute viral encephalomyelitis.

Semliki Forest Virus (SFV) causes a more severe acute encephalomyelitis in B6 than in SJL mice despite similar T cell proliferation and antibody responses in these two strains. To determine the immunological mechanisms that may contribute to this difference, CNS tissues from SFV-infected B6 and SJL mice were analyzed for viral replication, inflammatory responses and cytokine production, by semiquantitative reverse transcriptase-PCR and immunohistochemistry. Although initially similar on day 2 p.i., SFV replicated to higher viral titers in B6 than SJL mice on days 4 and 7 p.i. Infectious virus was cleared from both strains by day 10 p.i. There were no differences in numbers of CD4+, CD8+ or MHC class I and II+ inflammatory cells at any time point. Higher levels of IL-4 mRNA, lower levels of TNF-alpha, IL-6, IL-1 beta and IL-2 mRNAs and lower IL-2+ and IFN-gamma+ cells were found in B6. These findings suggest that despite comparable immune responses, different patterns of cytokine production correlated with higher levels of virus in the brains and more severe clinical disease in B6, and more efficient clearance of virus and less severe disease in SJL mice.

Human leukocyte antigens and cytokine expression in cerebral inflammatory demyelinative lesions of X-linked adrenoleukodystrophy and multiple sclerosis.

The two most common forms of X-linked adrenoleukodystrophy (X-ALD) are the cerebral forms (CER) with an inflammatory demyelinating reaction that resembles multiple sclerosis, and adrenomyeloneuropathy (AMN) which involves primarily the spinal cord and in which the inflammatory reaction is mild or absent. We found no significant association between the childhood cerebral form (CCER) or AMN and the human leukocyte (HLA) class I and Class II antigens including the class II DR2 haplotypes associated with multiple sclerosis. Inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1 beta, interleukin-4, interleukin-6 and interferon-gamma) gene expression was increased in multiple sclerosis brain lesions, as has been reported previously, but much less so in CER brain lesions. These findings suggest that the pathogenesis of the inflammatory response in X-ALD differs from that in multiple sclerosis.

Transneuronal transport of herpes simplex virus from the cervical vagus to brain neurons with axonal inputs to central vagal sensory nuclei in the rat.

The recent introduction of live viruses as intra-axonal tracing agents has raised questions concerning which central neurons are transneuronally labelled after application of the virus to peripheral organs or peripheral nerves. Since the central connections of the vagus nerve have been well described using conventional neuronal tracing agents, we chose to inject Herpes Simplex Virus Type 1 into the cervical vagus of the rat. After survival times of up to 3 days the rat brains were processed immunohistochemically using a polyclonal antiserum against herpes simplex virus. Two days after injection of the virus we observed viral antigen in the area postrema and in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (dorsal vagal complex), principally ipsilaterally. At this survival time the viral antigen in the dorsal vagal complex was largely confined to glial cells. After 3 days the viral antigen was localized both in glia and in nerve cells within the dorsal vagal complex and in brain regions previously demonstrated, using conventional tracing procedures, to contain neurons with axonal projections to the dorsal vagal complex. This was true for medullary, pontine, midbrain and hypothalamic regions and for telencephalic regions including the amygdala, the bed nucleus of the stria terminalis, and the insular and medial frontal cortices. Many of the nerve cells containing viral antigen were displayed in a Golgi-like manner, with excellent visualization of the dendritic tree. Axonal processes, in contrast, were not visualized. We used co-localization studies to confirm previous findings concerning monoamine neurotransmitter-related antigens present in medullary and pontine neurons projecting to the dorsal vagal complex. After 3 days there were many Herpes Simplex Virus Type 1-containing glial cells along the intra-medullary course of the vagal rootlets. However, no viral antigen was found in brain regions containing neurons whose axons pass through the region of glial cell-labelled rootlets. Glial cells containing viral antigen were particularly numerous in brain regions known to receive an input from neurons in the area postrema and the dorsal vagal complex. Taken together with our observation concerning the early appearance of viral antigen within glial cells in the dorsal vagal complex, this suggests that when the virus reaches the axon terminal portion it is transferred to nearby glial cells and possibly enters central neurons by way of these structures.

Renal sympathetic preganglionic neurons demonstrated by herpes simplex virus transneuronal labelling in the rabbit: close apposition of neuropeptide Y-immunoreactive terminals.

Renal sympathetic preganglionic neurons in the spinal cord of rabbits were transneuronally retrogradely labelled by injection of Herpes simplex virus type 1 into the renal nerve and immunohistochemical demonstration of viral antigen. The morphology of the labelled neurons was examined, particularly with respect to the shape and extent of their dendritic trees. Double-labelling immunohistochemical studies were performed to determine the relationship of neuropeptide Y-immunoreactive axons to virus-labelled perikarya and dendrites. The shape of the renal sympathetic preganglionic neurons differed according to whether the neurons were located in the intermediolateral cell column or in other sympathetic areas. The neurons in the intermediolateral cell column had very long dendrites, extending in the rostrocaudal and mediolateral directions. The medially oriented processes extended towards and beyond the central canal. The laterally oriented dendritic processes projected within the dorsolateral funiculus, towards the edge of the spinal cord. Neuropeptide Y-immunoreactive fibres were concentrated in regions containing renal sympathetic preganglionic neurons of the spinal segments examined (T7-L2). Immunoreactive varicose terminals were closely opposed to individual preganglionic neurons, especially to the dendritic processes of these neurons. Our findings indicate that neurotransmitter candidates such as neuropeptide Y are likely to influence renal preganglionic neurons by an input to dendritic processes at some distance from the perikarya. Electrophysiological and other functional studies utilizing applications of neurotransmitter candidates onto these neurons should take this into account.

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.

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.

Cytomegalovirus viral load monitoring after allogeneic bone marrow transplantation in patients receiving antiviral prophylaxis.

Cytomegalovirus viral load measurement is a powerful new tool for monitoring of CMV disease; however, the optimal strategy for use is unknown. Weekly plasma CMV viral loads and CMV-related outcomes were monitored in 46 consecutive allogeneic bone marrow transplantation (BMT) recipients receiving standardised antiviral prophylaxis. A total of 412 CMV viral loads were quantitated in the first 100 days post transplantation with 77 positive samples (19%) in 20 patients (43%). No patient with all negative CMV viral load results developed CMV disease. Two of three patients with highly positive CMV viral loads (first positive < or =30 days post transplant, maximum viral load > or =5000 copies/ml, and > or =50% of samples positive) developed CMV disease. A total of 17 patients with positive CMV viral loads, who did not meet the criteria for highly positive, did not develop CMV disease. CMV viral load detection was higher in recipients who were CMV sero-positive. In conclusion, CMV disease did not occur in the setting of a persistently negative CMV viral load. A positive CMV viral load result occurred commonly after allogeneic BMT, even in patients receiving antiviral prophylaxis.

Clinical, microbiological, and economic benefit of a change in antibiotic prophylaxis for cardiac surgery.

Vancomycin and rifampicin replaced cephazolin as antibiotic prophylaxis for coronary artery bypass surgery at our institution. Following this intervention, there was a significant decrease (P < .001) in the surgical-site infection rate from 10.5 (95% confidence interval, 8.2 to 13.3) to 4.9 (95% confidence interval, 3.2 to 7.1) infections per 100 procedures. An estimated $576,655 (Australian) was saved between two 12-month periods.

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.

Chlamydia pneumoniae in HIV-infected patients and controls assessed by a novel whole blood interferon-gamma assay, serology and PCR.

Chlamydia pneumoniae seropositivity is associated with cardiovascular disease and HIV infection. Cell-mediated immune responses are important for control of C. pneumoniae, and such responses may be impaired in HIV-infected patients. An assay for detection of interferon (IFN)-gamma in whole blood stimulated with C. pneumoniae antigen was developed and studied in HIV-infected patients and uninfected controls. Among 34 HIV-infected patients, none had an IFN-gamma response to C. pneumoniae antigen, compared with five of 32 healthy controls (p < 0.001). Fewer HIV-infected individuals elicited a serum IgG response when tested with a commercial enzyme immunoassay (p 0.009), but this was not so for serum IgA (p 0.12). Additionally, the IFN-gamma and antibody assays showed a trend towards a bivariate response in normal controls. This indicates that cellular and antibody responses against C. pneumoniae may be mutually exclusive, with potential implications for the role of this organism in the genesis of cardiovascular disease in both immunocompetent and HIV-infected populations.