Recent Advances in Biomedical Research: Targeting HIV Latency, Unraveling Cortico-Cortical Pain Modulation, and Enhancing Gut Barrier Integrity

PKC Agonists as Latency-Reversing Agents in HIV

Latent HIV reservoirs persist in individuals receiving combination antiretroviral therapy (ART), posing a significant barrier to achieving a cure. One promising strategy to eliminate these reservoirs is the “shock and kill” approach, which involves using latency-reversing agents (LRAs) to induce expression of latent HIV followed by immune-mediated clearance. Among the most effective LRAs are PKC agonists.

However, non-selective PKC agonists (e.g., prostratin, bryostatin, and certain DAG-lactones) activate a broad range of PKC isoforms and are associated with significant toxicity. Preclinical studies have highlighted severe adverse effects—most notably widespread platelet activation that may lead to disseminated intravascular coagulation (DIC). To overcome these limitations, recent work has focused on identifying and selectively activating the novel PKC isoforms, particularly PKC-ε and PKC-η, which are abundantly expressed in CD4⁺ T cells but not in platelets.

Improving PKC Agonist Selectivity

Selective activation of novel PKC isoforms can reduce the off-target activation of platelets and thus mitigate severe toxicity. Using structure-based drug design, researchers have developed synthetic diacylglycerol (DAG)-lactone derivatives that demonstrate enhanced selectivity toward PKC-ε. For example, the novel compounds C-232A and its improved derivative C-233 have been designed to preferentially target PKC-ε. This specificity is critical because it allows the induction of HIV expression in CD4⁺ T cells with significantly lower toxicity in platelets.

Below is a table summarizing the selectivity of common PKC inhibitors measured by their half-maximal inhibitory concentration (IC50) values across various PKC isoforms:

Table 1. IC50 values demonstrating the inhibitory profile of PKC inhibitors on classical and novel PKC isoforms. (Data adapted from study data.)

Further evaluation focused on the translocation properties of novel synthetic PKC agonists. When assessed in A549 cells engineered to express tGFP-fused PKC isoforms, the compounds demonstrated distinct profiles of potency and selectivity. The following table compares the effective concentration (EC50) for membrane translocation of PKC isoforms upon treatment with C-232A and the improved selective agent C-233:

Table 2. EC50 values for PKC isoform translocation in A549-tGFP cells, showing a marked improvement in selectivity for PKC-ε with C-233.

Importantly, ex vivo studies using total CD4⁺ T cells from ART-suppressed individuals demonstrated that C-233 robustly induced both HIV RNA and viral protein (p24) expression at doses that produced minimal platelet activation. In vitro assays measuring both T-cell activation (via upregulation of the early activation marker CD69) and platelet activation (assessed by CD62P expression) in whole blood have shown that C-233 is approximately 5-fold more potent for T-cell activation compared to its effect on platelets. These findings provide a strong rationale for further clinical investigation of selective, PKC-ε–targeted compounds as latency-reversing agents with improved safety profiles.

Cortico-Cortical Facilitation of Nociceptive Processing: The Retrosplenial–Anterior Cingulate Cortex Pathway

Chronic and acute pain have long been studied with an emphasis on subcortical networks; however, recent work has revealed that cortico-cortical circuits also play significant roles in modulating nociception. A novel study has provided compelling evidence for a direct excitatory projection from the retrosplenial cortex (RSC) to the anterior cingulate cortex (ACC), two brain regions known for their roles in memory and pain perception, respectively.

Anatomy and Physiology of the RSC-ACC Projection

Using advanced whole-brain imaging techniques, such as Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR), researchers have delineated the anatomical connectivity between the RSC and ACC in mouse models. Trans-monosynaptic retrograde tracing demonstrated robust labeling of neurons in the RSC that project directly to the ACC. These anatomical findings were corroborated by:

• Multichannel Field Potential Recordings: In vitro field potential recordings in brain slices showed that electrical stimulation in the RSC elicited robust excitatory postsynaptic potentials in the ACC. The amplitude of these responses decreased as a function of distance from the stimulation site, indicating a direct, gradient-dependent transmission.

• Two-Photon Calcium Imaging: Stimulation of the RSC, either via electrical pulses or through the local application of glutamate, significantly increased intracellular Ca²⁺ levels in ACC pyramidal neurons. These calcium transients were closely associated with the synaptic activation of postsynaptic receptors.

• Pharmacological Characterization: The excitatory synaptic transmission from the RSC to the ACC was found to be predominantly mediated by AMPA receptors, as evidenced by the significant reduction in evoked excitatory responses upon application of selective AMPA receptor antagonists (such as GYKI 53655). Blockade of NMDA or kainate receptors had minimal effects on these synaptic currents.

Implications for Nociceptive Processing

Behavioral experiments using optogenetic and chemogenetic techniques have provided evidence that activation of the RSC-ACC pathway enhances both mechanical and thermal nociceptive responses in mice. Notably, targeted stimulation of this pathway decreased hindpaw withdrawal thresholds in mechanical (von Frey) and hot plate tests, indicating that the RSC-ACC projection exerts a facilitatory influence on pain perception at the supraspinal level. Interestingly, such activation did not significantly alter outputs in spinal-level nociceptive reflexes (e.g., tail-flick test), suggesting its effect is primarily cortical.

Enhancing Gut Barrier Integrity with Fecal Microbiota Transplantation and Akkermansia muciniphila

The gut barrier is essential for protecting the host against pathogenic invasion and systemic inflammation. Antibiotic-induced dysbiosis can compromise this barrier, increasing susceptibility to infections like those caused by enterotoxigenic Escherichia coli (ETEC) K88. Fecal microbiota transplantation (FMT) has emerged as a promising therapeutic strategy for restoring microbial balance and enhancing gut barrier function.

FMT in Microbiome-Disordered Models

Experimental studies in antibiotic-induced microbiome-disordered (AIMD) piglets infected with ETEC K88 have shown that FMT:

• Improves growth performance by increasing average daily weight gain.
• Reduces clinical symptoms such as diarrhea.
• Lowers ETEC colonization in the jejunal and colonic mucosa.

Histological examinations revealed that FMT-treated animals exhibited improved intestinal morphology with restored villi and microvilli integrity. Moreover, there was a significant recovery in the expression of tight junction proteins (including ZO-1, claudin, and occludin) as well as adherens junction proteins (β-catenin and E-cadherin). Immunofluorescence analysis further confirmed enhanced expression of Mucin 2 (MUC2), a critical mucus component, in the FMT group.

Identification of Key Microbial Strains

Using 16S rDNA gene sequencing and LEfSe (linear discriminant analysis effect size) analysis, two bacterial strains emerged as key players in the protective effects observed following FMT:

• Akkermansia muciniphila: This mucin-degrading bacterium has been shown to support the proliferation and differentiation of intestinal stem cells via the activation of the Wnt/β-catenin signaling pathway. Studies using both in vivo murine models and in vitro intestinal organoid systems demonstrated that A. muciniphila effectively protects against ETEC-induced epithelial injury.

• Bacteroides fragilis: Although beneficial, comparative data suggest that B. fragilis is less potent than A. muciniphila in modulating the critical signaling pathways (e.g., Wnt/β-catenin) required to strengthen the gut barrier.

Supplementary data from these experiments include significant changes in gene expression levels and morphological improvements following treatment with these strains, reinforcing their potential role as a targeted probiotic therapy.

Conclusion and Future Perspectives

The three research areas highlighted in this review exemplify the innovative strategies being developed to address complex biomedical challenges:

1. HIV Latency Reversal: By leveraging structure-based drug design, researchers have developed selective PKC agonists (such as C-233) that preferentially activate PKC-ε—thereby effectively reversing HIV latency with reduced adverse effects, particularly minimizing platelet activation.

2. Nociceptive Modulation: The identification of a direct excitatory glutamatergic pathway from the RSC to the ACC offers new insights into the supraspinal mechanisms involved in pain perception. This cortico-cortical circuit may serve as a potential target for the modulation of chronic pain without affecting spinal nociceptive reflexes.

3. Gut Barrier Restoration: FMT, coupled with the targeted application of beneficial microbes like Akkermansia muciniphila, has demonstrated significant efficacy in restoring gut barrier function after antibiotic-induced dysbiosis and pathogenic challenge. Through modulation of the Wnt/β-catenin signaling pathway, A. muciniphila enhances intestinal epithelial regeneration and reduces inflammation.

Future research should focus on translating these findings into clinical interventions. Further optimization of PKC agonist selectivity, advanced modulation of pain circuits, and precise microbial therapies promise to reshape treatment landscapes in HIV cure strategies, pain management, and gastrointestinal health.

Frequently Asked Questions (FAQ)

What are PKC agonists and why are they important in HIV research?
Protein kinase C (PKC) agonists are compounds that activate specific PKC isoforms. They are important in HIV research because they can induce the expression of latent HIV in CD4⁺ T cells. By selectively targeting isoforms like PKC-ε, it is possible to reverse latency while minimizing unwanted side effects such as platelet activation.

How does the selective activation of PKC-ε improve tolerability?
Platelets express several PKC isoforms; however, PKC-ε is less expressed in platelets but abundant in CD4⁺ T cells. By designing agonists that selectively activate PKC-ε, such as C-233, researchers can achieve T-cell activation and HIV expression with reduced platelet activation, thereby lowering the risk of coagulation-related toxicities.

What is the significance of the RSC-ACC pathway in pain modulation?
The retrosplenial cortex (RSC) to anterior cingulate cortex (ACC) pathway represents a direct excitatory connection that modulates nociceptive processing. Activation of this pathway increases intracellular calcium in ACC neurons via AMPA receptor-mediated transmission, which facilitates the perception of pain at a supraspinal level.

What role does Akkermansia muciniphila play in gut barrier function?
Akkermansia muciniphila is a beneficial bacterium that enhances gut barrier integrity by promoting the proliferation and differentiation of intestinal stem cells. It activates the Wnt/β-catenin signaling pathway, leading to improved expression of mucins and tight junction proteins, which protect the gut from pathogen invasion.

What future directions are anticipated based on these findings?
Future research may focus on:

• Clinical development and refinement of selective PKC agonists for HIV latency reversal with reduced toxicity.
• Exploring the therapeutic potential of modulating the RSC-ACC pathway to manage chronic pain.
• Advancing microbiome-based therapies, particularly the use of muciniphila, to restore gut barrier function in patients suffering from dysbiosis-related disorders.

References

1. Irrinki, A., et al. (2025). Activating PKC-ε induces HIV expression with improved tolerability. PLOS Pathogens. https://doi.org/10.1371/journal.ppat.1012874

2. Hao, S., et al. (2025). Supraspinal facilitation of painful stimuli by glutamatergic innervation from the retrosplenial to the anterior cingulate cortex. PLOS Biology. https://doi.org/10.1371/journal.pbio.3003011

3. Ma, X., et al. (2023). Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway. eLife. https://doi.org/10.7554/eLife.92906