Cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ −150 °C or can be stored for a specified time at −80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions. We identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ −150 °C. However, there were 18 genes identified to be different when PBMCs were stored at −80 °C but did not change when stored < −150 °C. A correlation was also found between the expressions of 2′–5′- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ −150 °C as compared to −80 °C. Not only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between −80 °C and −150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ −150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.

Study of a clinic case reveals that alpha-1-antitrypsin (AAT) deficiency is related to CD4+ T cell count decline and AIDS progression, suggesting that AAT might be an endogenous inhibitor of HIV/AIDS. Previous study shows that AAT inhibits HIV-1 replication in infected host cells and the C-terminus fragment of AAT, VIRIP, interferes with HIV-1 infection. However, it is still unclear whether and how intact AAT inhibits HIV-1 infection. It is also unknown what the mechanism of AAT is and which critical step(s) are involved. In the present study, the C-terminus of AAT (C) was synthesized. C terminus-truncated AAT (ΔAAT) was also prepared by digesting AAT with metalloproteinase. Primary CD4+ T cells were then co-cultured with HIV-1 with the presence or absence of AAT/C/ΔAAT to detect cis-infection of HIV-1. The interaction between AAT/C/ΔAAT and gp120/gp41 was also measured. Meanwhile, HIV-1 reverse transcriptase activity and viral DNA integration were also detected in these lymphocytes. The results demonstrated that AAT and C, not ΔAAT, inhibited HIV-1 entry by directly interacting with gp41. Meanwhile, AAT, C and ΔAAT could not directly interfere with the steps of viral RNA reverse transcription and viral DNA integration. AAT inhibits HIV-1 entry by directly interacting with gp41 through its C-terminus and thereby inhibits HIV-1 infection.

In α1-antitrypsin–deficient HIV patients, an accelerated decline of CD4+ T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4+ T cells. Thus, primary CD4+ T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4+ T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4+ T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4+ T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1–mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome–lysosome–cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.