In this study we selected Synbiotic 2000® based on its documented activity in protecting hospitalized patients from infectious complications [35
] and in reducing circulating inflammatory cytokines and LPS [31
]. While the first part of our hypothesis, that probiotic species would be enhanced in the gut, was supported, the proposed drop in microbial translocation and immune activation was not observed. This could be interpreted as meaning that the chosen probiotic bacteria are intrinsically unable to exert the hypothesized downstream effects, that is, reduction of microbial translocation and immune activation. Conversely, our data raise the question of how much colonization, in terms of magnitude and duration, would be required to generate the hypothesized subsequent effects if they were to occur at all. To tackle this issue it seems necessary to design future studies of this type into two phases, the first being the testing of multiple formulations with variable dosing and time frames to establish optimal conditions for achieving substantial and reproducible enhancement in probiotic bacteria in the gut of HIV patients. A key issue to grapple with in this first phase lies in the fact that there is an enormous array of possible formulations to choose from. This is a reflection of the large diversity of probiotic species, the plethora of specific strains with unique properties, and the formulation of mixtures containing multiple probiotic species. With the addition of prebiotics as an additional variable, it becomes clear why the formulations in intervention studies vary so widely. The good news with this situation is that there is a rich, diverse palette available to the clinical scientist, while the negative aspect is that a formulation with bacteria lacking the desired properties may be chosen. As comprehensive studies on intestinal microbiota alterations in HIV infection become available, it may be possible to tailor interventions appropriately. These challenges aside, after establishing optimal formulations in phase one, the most promising strategies can then be tested in phase two to determine whether there are beneficial effects on gut and immune function.
The relevance of the modest changes in activation phenotypes (Table and Figure ) is not clear. The significant p-values were borderline and may have been influenced by the use of multiple comparisons. In addition, the difference in mean changes was small in both cases of statistical significance. For example, the phenotype consisting of CD4+ cells expressing HLA-DR but not CD38 or PD-1 is a very small population which changed a small amount (from 1.9 to 2.1
% of CD4+ T-cells). It would seem that if the modest change in this population in the synbiotic arm was a result of the synbiotic intervention then a correlation between this phenotype and stool levels of supplemented bacteria would be evident, which was not the case. Likewise, the other treatment-associated change, CD38 antibodies bound per CD8+ T-cell, did not correlate with stool probiotic bacteria either. Furthermore, the major activation phenotypes that we would have predicted to change in response to the synbiotic intervention (CD4+ and CD8+ T-cells double positive for HLA-DR and CD38) remained stable. Thus, the significance of the observed small shifts in T-cell subsets, and whether they actually resulted from experimental intervention remains in question.
Measuring microbial translocation requires accurate measurement of microbial constituents such as LPS or bacterial DNA in circulation. We chose to measure bacterial ribosomal 16S levels in order to take advantage of the reproducible and quantitative nature of real-time PCR. With this approach it is important to bear in mind that what is being measured is the presence of a highly conserved DNA sequence, 204
bp in our case, and not necessarily the presence of entire bacterial genomes or intact bacteria in circulation. Other studies have reported elevated plasma LPS and bacterial r16S concentration in HIV patients compared to HIV seronegatives [11
]. While our study cohort was entirely HIV seropositive, we also measured r16S levels in the plasma of 10 volunteers at low-risk for HIV infection and found the average concentration to be reduced by 50% (data not shown). Related to bacterial translocation is the emerging surrogate marker sCD14 which is released from monocytes in response to LPS exposure, and in HIV patients is correlated with disease progression [41
]. Our primary endpoint of microbial translocation was therefore assessed both directly via bacteria r16S and indirectly using the host factor sCD14. Data from these two methods support the conclusion that there was no change in microbial translocation. Finally, liver function may be relevant in studies of microbial translocation as the liver may remove incoming bacteria from the portal circulation to varying extents and the inclusion of markers of liver function therefore advisable. In this study, CRP, made by the liver as an acute phase protein, was also unaltered by the synbiotic.
Recent studies by other groups support the concept that pre- and probiotics may have positive impacts on CD4+ T-cell function in HIV infection [42
]. For instance, Trois et. al. tested a probiotic formula containing Bifidobacterium bifidum
and Streptococcus thermophilus
, administered daily for 2
months to pediatric HIV patients on antiretroviral therapy, and observed a significant rise in blood CD4+ T-cell count (+118 cells/μl) compared to placebo (−42 cells/μl) [43
]. A significant rise in blood CD4 T-cell count was also documented in separate studies of HIV infection carried out in Nigeria and Tanzania using a probiotic yogurt containing either L. rhamnosus
] or L. rhamnosus
GR-1 plus L. reuteri
]. Interestingly, a subsequent longer term 25
week study using L. rhamnosus
GR-1 plus L. reuteri
RC-14 did not reveal a CD4+ T-cell modulating effect, possibly due to the use of an encapsulated rather than a yogurt formulation [46
]. Importantly, studies performed thus far have demonstrated safety in the administration of probiotics to HIV patients.
With regard to prebiotics, a pilot randomized 12
week trial of an oligosaccharide mixture in HAART-naïve HIV patients revealed reduced activation of peripheral CD4+ but not CD8+ T-cells (as measured by CD25 expression), but no change in blood CD4 T-cell count [47
]. One explanation of these findings is that a drop in CD4+ T-cell activation may precede a subsequent, slower rise in CD4 count. In fact, in patients initiating HAART with CD4+ T-cell counts greater than 200 cells/μl, CD38 expression on peripheral CD4+ T-cells drops significantly prior to a rise in blood CD4 T-cell count [48
]. Further, this pattern does not hold true for CD8+ T-cells, which lose CD38 expression more slowly after initiation of HAART. Given this pattern it seems likely that an additional incremental reconstitution of CD4+ T-cells in response to a pre- or probiotic intervention may be preceded by an initial drop in CD38 expression on CD4+ T-cells.
The present study had several limitations. First, because it was a pilot study the number of subjects enrolled was small and we focused on patients taking antiretroviral drugs. Had we been able to include patients with AIDS-defining characteristics, a group previously shown to have higher levels of immune activation and bacterial translocation than those taking antiretroviral drugs [11
], the effects of the synbiotic may have been more clear. Another issue is that there was not a mechanism to reliably assess compliance, other than the subjects’ personal record keeping on ingesting the formulations which consistently indicated full compliance. Compliance seemed especially relevant given the unusual texture of the interventions and their tendency to produce intestinal gas, especially during the first week. A third limitation was the placebo being made up of the fiber component of the synbiotic. While this formulation did make it possible to implement double-blinded assignments, the fact that fiber alone can exert biological effects renders it a less than optimal placebo. Nevertheless, any differences between synbiotic and fiber-only can be taken as due to the probiotic component of the synbiotic. An additional limitation is that the measurement of circulating bacterial ribosomal 16S levels by qPCR does not yield information on the specific types of bacteria that are being translocated. The inclusion of sequencing to yield this information in future studies would be informative. Lastly, it was beyond the scope of this study to obtain intestinal mucosal biopsies. In response to pre-, pro- and synbiotics, some of the earliest and most important changes in immune cell phenotype and function may occur in the intestinal mucosa, and in this study we would have missed them.