Development of Droplet Digital PCR-Based Assays to Quantify HIV Proviral and Integrated DNA in Brain Tissues from Viremic Individuals with Encephalitis and Virally Suppressed Aviremic Individuals.

Although combination antiretroviral therapy (cART) can suppress the replication of HIV, the virus persists and rebounds when treatment is stopped. To find a cure that can eradicate latent reservoir, a method should be able to quantify the lingering HIV. Unlike other digital PCR technologies, droplet digital PCR (ddPCR), provides absolute quantification of target DNA molecules using fluorescent dually labeled probes by massively partitioning the sample into droplets. ddPCR enables exquisitely sensitive detection and quantification of viral DNA from very limiting clinical samples, including brain tissues. We developed and optimized duplex ddPCR assays for the detection and quantification of HIV proviral DNA and integrated DNA in the brain of HIV-1-infected patients. We have applied these approaches to successfully analyze 77 human brain tissues obtained from 27 HIV-1-infected individuals, either fully virally suppressed or with encephalitis, and were able to quantify low levels of viral DNA. Further developments and advancement of digital PCR technology is promising to aid in accurate quantification and characterization of the persistent HIV reservoir. IMPORTANCE We developed ddPCR assays to quantitatively measure HIV DNA and used this ddPCR assays to detect and quantitatively measure HIV DNA in the archived brain tissues from HIV patients. The tissue viral loads assessed by ddPCR was highly correlative with those assessed by qPCR. HIV DNA in the brain was detected more frequently by ddPCR than by qPCR. ddPCR also showed higher sensitivity than qPCR since ddPCR detected HIV DNA signals in some tissues from virally suppressed individuals while qPCR could not.

Phenotypic and Functional Characterization of Circulatory, Splenic, and Hepatic NK Cells in Simian Immunodeficiency Virus-Controlling Macaques.

NK cells are key components of the immune system because of their rapid response potential and their ability to mediate cytotoxic and immunomodulatory functions. Additionally, NK cells have recently been shown to persist for long periods in vivo and to have the capacity to establish immunologic memory. In the current study, we assessed the phenotype and function of circulatory and tissue-resident NK cells in a unique cohort of SIV-controlling rhesus macaques that maintained low to undetectable levels of viremia in the chronic phase of infection. By contrasting NK responses of these macaques with those observed in SIV-noncontrolling and uninfected macaques, we aimed to identify markers and activities of NK subpopulations associated with disease control. We show in this article that most differences among NK cells of the three groups of macaques were observed in tissue-resident cells. Although SIV infection resulted in NK cell dysfunction, double-negative NK cells and those expressing CXCR3, NKG2D, and IL-18Rα were associated with viremia control, as was Ab-dependent cytotoxic function. Our results suggest several novel targets for therapeutic intervention.

Tyrosine 705 Phosphorylation of STAT3 Is Associated with Phenotype Severity in TGFß1 Transgenic Mice.

Transforming growth factor beta 1 (TGFß1) is a key player in skeletal muscle degenerative and regenerative processes. We previously showed that conditionally overexpressing TGFß1 in skeletal muscles caused myofiber atrophy and endomysial fibrosis in mice. However, the disease severity varied significantly among individual mice. While 40% of mice developed severe muscle pathology and lost body weight within 2 weeks of TGFß1 transgene induction in muscles, the rest showed milder or no phenotype. This study aims at determining whether signal transducer and activator of transcription 3 (STAT3) plays a role in the phenotypic difference and whether it can be activated by TGFß1 directly in muscle cells. Our results show that while total STAT3 was not differentially expressed between the two groups of mice, there was significantly higher pSTAT3 (Tyr705) in the muscles of the mice with severe phenotype. Immunohistochemistry showed that pSTAT3 (Tyr705) was localized in approximately 50% of the nuclei of the muscles. We further showed that TGFß1 induced Tyr705 phosphorylation of STAT3 in C2C12 cells within 30 minutes of treatment while total STAT3 was not affected. Our findings suggest that TGFß1 alone can induce Tyr705 phosphorylation of STAT3 in skeletal muscle cells and contribute to disease severity in transgenic TGFß1 mice.

Conditional expression of TGF-ß1 in skeletal muscles causes endomysial fibrosis and myofibers atrophy.

To study the effects of transforming growth factor beta 1 (TGF-ß1) on fibrosis and failure of regeneration of skeletal muscles, we generated a tet-repressible muscle-specific TGF-ß1 transgenic mouse in which expression of TGF-ß1 is controlled by oral doxycycline. The mice developed muscle weakness and atrophy after TGF-ß1 over-expression. We defined the group of mice that showed phenotype within 2 weeks as early onset (EO) and the rest as late onset (LO), which allowed us to further examine phenotypic differences between the groups. While only mice in the EO group showed significant muscle weakness, pathological changes including endomysial fibrosis and smaller myofibers were observed in both groups at two weeks after the TGF-ß1 was over-expressed. In addition, the size of the myofibers and collagen accumulation were significantly different between the two groups. The amount of latent and active TGF-ß1 in the muscle and circulation were significantly higher in the EO group compared to the LO or control groups. The up-regulation of the latent TGF-ß1 indicated that endogenous TGF-ß1 was induced by the expression of the TGF-ß1 transgene. Our studies showed that the primary effects of TGF-ß1 over-expression in skeletal muscles are muscle wasting and endomysial fibrosis. In addition, the severity of the pathology is associated with the total amount of TGF-ß1 and the expression of endogenous TGF-ß1. The findings suggest that an auto-feedback loop of TGF-ß1 may contribute to the severity of phenotypes.