Phage therapy to control multidrug-resistant Pseudomonas aeruginosa skin infections: in vitro and ex vivo experiments

The main goal of this study was to evaluate the efficiency of phage therapy against one of the most common multidrug-resistant (MDR) agents of skin infections, Pseudomonas aeruginosa. A phage suspension [10⁸?plaque-forming units (PFU)?mL^?1] was obtained using the clinical strain P. aeruginosa 709 as the host. The ability of the phage to inactivate P. aeruginosa was evaluated in vitro and ex vivo (human skin), using a multiplicity of infection (MOI) of 0.5 to 50. In the presence of the phage, the density of P. aeruginosa 709 [10⁵?colony-forming units (CFU)?mL^?1] in the human skin decreased by 4 logs after 2?h of incubation. The application of a second dose of phage did not increase the efficiency of the therapy. This study indicates that the topical application of phage PA709 efficiently inactivates MDR P. aeruginosa 709. The high efficiency in the inactivation of MDR P. aeruginosa 709, its considerable host range (infection of 30?% of the P. aeruginosa isolates) and its high stability in buffer and ex vivo human skin make this phage very promising for the treatment of P. aeruginosa skin infections. The phage–bacteria interactions were examined in vitro and in ex vivo in order to provide a basis for the selection of the most suitable protocol for subsequent in vivo experiments.     

HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model

Infection and dissemination of human immunodeficiency virus (HIV)-1 through the female body after vaginal intercourse depends on the activation/differentiation status of mucosal CD4 T cells. In this study, we investigated this status and the susceptibility to HIV-1 infection of human cervico-vaginal tissue ex vivo. We found that virtually all T cells are of the effector memory phenotype with broad CC chemokine receptor 5 (CCR5) expression. As it does in vivo, human cervico-vaginal tissue ex vivo preferentially supports the productive infection of R5 HIV-1 rather than that of X4 HIV-1 in spite of the broad expression of CXC chemokine receptor 4 (CXCR4). X4 HIV-1 replicated only in the few tissues that were enriched in CD27⁺CD28⁺ effector memory CD4 T cells. Productive infection of R5 HIV-1 occurred preferentially in activated CD38⁺CD4 T cells and was followed by a similar activation of HIV-1-uninfected (bystander) CD4 T cells that may amplify viral infection. These results provide new insights into the dependence of HIV-1 infection and dissemination on the activation/differentiation of cervico-vaginal lymphocytes.     

Differentiation of human immunodeficiency virus type 1 (HIV-1) infections with HIV-2-cross-reacting antibody from mixed infections with HIV-1 and HIV-2 by serological absorption test.

The interpretation of dual seroreactivity with human immunodeficiency virus type 1 (HIV-1) and HIV-2 in blood samples is a serious problem facing AIDS researchers worldwide. Some samples of sera from HIV-1-infected patients showed a serological cross-reaction with HIV-2, causing confusion regarding the serodiagnosis. Therefore, we tried to differentiate these serum samples from those containing real mixed infections with both types of virus. Sera from patients with HIV-1 infections with HIV-2 cross-reacting antibody in Japan were distinguished from sera from patients with mixed infections with HIV-1 and HIV-2 in West Africa by our serological cross-absorption test, which proved to be highly specific and useful for serodiagnosis.     

CD163 overexpression using a macrophage-directed gene therapy approach improves wound healing in ex vivo and in vivo human skin models

Large tissue damage or wounds cause serious comorbidities and represent a major burden for patients, families, and health systems. Due to the pivotal role of immune cells in the proper resolution of inflammation and tissue repair, we focus our current study on the interaction of macrophages with skin cells, and specifically on the effects of CD163 gene induction in macrophages in wound healing. We hypothesize that the over-expression of the scavenger receptor gene CD163 in human macrophages would result in a more efficient wound healing process. Using 3D human wounded skin organotypic tissues, we observed that CD163 overexpression in THP-1 and human primary macrophages induced a more efficient re-epithelization when compared to control cells. Using human primary skin cells and an in vitro scratch assay we observed that CD163 overexpression in THP-1 macrophages promoted a more rapid and efficient wound healing process through a unique interaction with fibroblasts. The addition of CD163-blocking antibody, but not isotype control, blocked the efficient wound healing process induced by CD163 overexpression in macrophages. We found that the co-culture of skin cells and CD163 overexpressing macrophages reduced monocyte chemoattractant protein (MCP)-1 and enhanced tumor growth factor (TGF)-α, without altering interleukin (IL)-6 or TGF-β. Our findings show that CD163 induces a more efficient wound healing and seems to promote a wound milieu with a pro-resolution molecular profile. Our studies set the foundation to study this approach in in vivo clinically relevant settings to test its effects in wound healing processes such as acute major injuries, large surgeries, or chronic ulcers.     

Sustained in vivo gene delivery from agarose hydrogel prolongs nonviral gene expression in skin

Prolonging gene expression in skin using safe, nonviral gene delivery techniques could impact skin regeneration and wound healing, decrease infection, and potentially improve the success of tissue-engineered skin. To this end, an injectable, agarose-based delivery system was tested and shown to prolong nonviral gene expression in the skin. DNA was compacted with polylysine to improve DNA stability in the presence of nucleases. Up to 25 microg of compacted luciferase plasmid with or without agarose hydrogel was injected intradermally in rodents. Bioluminescence imaging was used for longitudinal, noninvasive monitoring of gene expression in vivo for 35 days. Injections of DNA in solution produced gene expression for only 5-7 days, whereas the sustained release of compacted DNA from the agarose system prolonged expression, with more than 500 pg (20% of day 1 levels) of luciferase per site for at least 35 days. Southern blotting confirmed that the agarose system extended DNA retention, with significant plasmid present through day 7, as compared with DNA in solution, which had detectable DNA only on day 1. Histology revealed that agarose invoked a wound-healing response through day 14. Tissue-engineering and wound-healing applications may benefit from the agarose gene delivery system.     

Cervico-vaginal tissue ex vivo as a model to study early events in HIV-1 infection

Vaginal intercourse remains the most prevalent route of infection of women. In spite of many efforts, the detailed mechanisms of HIV-1 transmission in the female lower genital tract remain largely unknown. With all the obvious restrictions on studying these mechanisms in humans, their understanding depends on the development of adequate experimental models. Isolated cell cultures do not faithfully reproduce important aspects of cell-cell interactions in living tissues and tissue responses to pathogens. Explants and other types of ex vivo tissue models serve as a bridge between cell culture and tissues in vivo. Herein, we discuss various cervico-vaginal tissue models and their use in studying HIV vaginal transmission and consider future directions of such studies.     

Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1

We investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells. Thus, HIV-1 alone is sufficient to deplete productively infected CD4+ T cells but is not sufficient to cause the death of uninfected or latently infected CD4+ T cells.     

Coming of age: reconstruction of heterosexual HIV-1 transmission in human ex vivo organ culture systems

Heterosexual transmission of human immunodeficiency virus-1 (HIV-1), from men to women, involves exposure to infectious HIV-1 in semen. Therefore, the cellular and molecular processes that underlie HIV-1 transmission are closely interconnected with fundamental principles of human reproductive biology. Human ex vivo organ culture systems allow experimental reconstruction of HIV-1 transmission, using human semen and premenopausal cervicovaginal mucosal tissue, with specific emphasis on the progression from exposure to development of primary HIV-1 infection. Clearly, an isolated piece of human tissue cannot duplicate the full complexity of events in natural infections, but with correct observation of conventional medical and ethical standards, there is no opportunity to study HIV-1 exposure and primary infection in young women. Human mucosal organ cultures allow direct study of HIV-1 infection in a reproducible format while retaining major elements of complexity and variability that typify community-based HIV-1 transmission. Experimental manipulation of human mucosal tissue both allows and requires acquisition of new insights into basic processes of human mucosal immunology. Expanding from the current foundations, we believe that human organ cultures will become increasingly prominent in experimental studies of HIV-1 transmission and continuing efforts to prevent HIV-1 infection at human mucosal surfaces.     

Natural antibodies to HIV-tat epitopes and expression of HIV-1 genes in vivo

The tat regulatory protein of HIV-1 was expressed as a fusion protein in E. coli and used as antigen to detect antibodies against HIV-tat (anti-tat) in the serum of HIV-1 infected children and adults. HIV-1-infected children showed a higher frequency (55%) of anti-tat than HIV-1-infected adults (36%). Anti-tat were present in only 15% (3/20) of acutely infected individuals. Forty percent (10/25) of individuals with prolonged HIV-1 infection but without antigen were anti-tat positive. Only 13% (3/23) of HIV-1-antibody-positive individuals with prolonged HIV-1 antigenemia were anti-tat positive and titers of anti-tat antibodies declined with time. Pepscan analysis identified the amino terminus of HIV-tat as the major antibody-binding site. Antibodies to HIV-tat occurred as a harbinger of HIV-1 antigen expression and disappeared thereafter, possibly reflecting the transience of HIV-tat expression. Because of the low antigenicity of HIV-tat, antibodies to this regulatory protein are not a reliable marker for either early HIV-1 infection or subsequent disease progression.     

Differences in replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) are not determined by long terminal repeats (LTR)

The growth properties of molecular clones of a highly cytopathic Zairian HIV1-NDK and prototype viruses were compared to correlate genetic variations with biological changes. The cloned HIV1-NDK retained the highly replicating cytopathic phenotype and formed larger syncytia than the prototype. One of the major differences in the alignment of the nucleotide sequence of the HIV1-NDK and HIV1-BRU prototypes was localized in the negative regulatory element (NRE) of the long terminal repeat (LTR). In a chloramphenicol acetyl transferase (CAT) assay, we failed to detect a significant difference between LTR promoter activity of the prototype and HIV1-NDK, suggesting that the LTR of both phenotypes had a similar function. The complete recombinant provirus DNA molecules bearing HIV1 LTR derived from one phenotype and the rest of the genomes from the other phenotype were constructed and transfected. The high cytopathogenicity of both the original and the chimeric viruses was correlated with the high speed of virus replication. Cytopathogenicity, morphology of syncytia, and replication kinetics of the recombinant viruses were determined by the functions coded within an internal part of HIV1 genome, covering the gag to env region, which were, however, not within LTR.     

Sequences within the feline immunodeficiency virus long terminal repeat that regulate gene expression and respond to activation by feline herpesvirus type 1.

We constructed a series of deletion mutants of feline immunodeficiency virus (FIV) long terminal repeat (LTR) to identify the regions that regulate gene expression and are responsive to trans-activation of FIV LTR by feline herpes-virus type 1 (FHV-1). We demonstrated that sequences between -124 and -79, and between -21 and -32 (relative to the cap site) are essential for gene expression of FIV in Felis catus whole fetus 4 (fcwf-4) cells. Further, we demonstrated that the sequence between -63 and -23 responds to trans-activation of FIV LTR by FHV-1 in fcwf-4 cells.     

Enhanced expression of cylooxygenase-2 by UV in aged human skin in vivo

Prostaglandins (PGs) induced by UV may play important roles in UV-induced inflammation, photocarcinogenesis, and photoaging processes in human skin. The age-related PGE2 production and cyclooxygenase-2 (COX-2) expression in the human skin in vivo remain unclear. The purpose of this study was to examine the influence of aging on UV-induced PGE2 production and COX-2 expression in human skin in vivo. We found that aged human skin produces higher amounts of PGE2 than young skin, when exposed to UV. The inductions of COX-2 mRNA and protein by UV in aged skin were higher than those in the young skin, whereas COX-1 mRNA expression remained unchanged. Aged human macrophage expressed higher amounts of PGE2 and COX-2 protein constitutively, and also induced these species after LPS treatment more so than young cells. Our data suggest that skin aging may increase susceptibility to the development of skin cancer and photoaging, by enhanced PGE2 and COX-2 expression due to UV in human skin in vivo.     

Gene expression profile of megakaryocytes from human cord blood CD34(+) cells ex vivo expanded by thrombopoietin

Previously, we investigated the process of megakaryocytopoiesis during ex vivo expansion of human cord blood (CB) CD34(+) cells using thrombopoietin (TPO) and found that megakaryocytopoiesis was closely associated with apoptosis. To understand megakaryocytopoiesis at the molecular level, we performed a microserial analysis of gene expression (microSAGE) in megakaryocytes (MKs) and nonmegakaryocytes (non-MKs) derived from human CB CD34(+) cells by ex vivo expansion using TPO, and a total of 38909 tags, representing 8976 unique genes, were identified. In MKs, many of the known genes, including coagulation factor VII, P-selectin (CD62P), pim-1, azurocidin, defensin, and CD48 were highly expressed; meanwhile, those genes encoding some small G proteins of the Ras family (Rab 7 and Rab 11A) and glutathione S transferase family (1, 4, A2, omega, and pi) showed lower expression levels in MKs. These gene expression profiles will be useful to understand megakaryocytopoiesis at the molecular level, including apoptosis and related signal transduction pathways.     

Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study

We have previously described an antiapoptotic steady-state gene expression profile in circulating human monocytes from asymptomatic viremic HIV(+) donors, but the mechanism associated with this apoptosis resistance remains to be fully elucidated. Here, we show that Rb1 activation is a dominant feature of apoptosis resistance in monocytes exposed to HIV-1 in vivo (as measured ex vivo) and in vitro. Monocytes from asymptomatic viremic HIV(+) individuals show a positive correlation between levels of hypophosphorylated (active) Rb1 and VL in conjunction with increases in other p53-inducible proteins associated with antiapoptosis regulation, such as p21 and PAI-1 (SERPINE1), when compared with circulating monocytes from uninfected donors. Monocytes exposed in vitro to HIV-1 R5 isolates but not X4 isolates showed lower caspase-3 activation after apoptosis induction, indicating a role for the CCR5 signaling pathway. Moreover, monocytes exposed to R5 HIV-1 or MIP-1 β induced Rb1 and p21 expression and an accumulation of autophagy markers, LC3 and Beclin. The inhibition of Rb1 activity in HIV-1 R5 viral-exposed monocytes using siRNA led to increased apoptosis sensitivity, thereby confirming a central role for Rb1 in the antiapoptotic phenotype. Our data identify Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes in association with protective autophagy and contributing to monocyte survival during immune activation and/or HIV-1 viremia.