Blockade Of Replication Research Articles

Mechanisms of Virus-Induced Airway Immunity Dysfunction in the Pathogenesis of COPD Disease, Progression, and Exacerbation

Chronic obstructive pulmonary disease (COPD) is the integrated form of chronic obstructive bronchitis and pulmonary emphysema, characterized by persistent small airway inflammation and progressive irreversible airflow limitation. COPD is characterized by acute pulmonary exacerbations and associated accelerated lung function decline, hospitalization, readmission and an increased risk of mortality, leading to huge social-economic burdens. Recent evidence suggests ~50% of COPD acute exacerbations are connected with a range of respiratory viral infections. Nevertheless, respiratory viral infections have been linked to the severity and frequency of exacerbations and virus-induced secondary bacterial infections often result in a synergistic decline of lung function and longer hospitalization. Here, we review current advances in understanding the cellular and molecular mechanisms underlying the pathogenesis of COPD and the increased susceptibility to virus-induced exacerbations and associated immune dysfunction in patients with COPD. The multiple immune regulators and inflammatory signaling pathways known to be involved in host-virus responses are discussed. As respiratory viruses primarily target airway epithelial cells, virus-induced inflammatory responses in airway epithelium are of particular focus. Targeting virus-induced inflammatory pathways in airway epithelial cells such as Toll like receptors (TLRs), interferons, inflammasomes, or direct blockade of virus entry and replication may represent attractive future therapeutic targets with improved efficacy. Elucidation of the cellular and molecular mechanisms of virus infections in COPD pathogenesis will undoubtedly facilitate the development of these potential novel therapies that may attenuate the relentless progression of this heterogeneous and complex disease and reduce morbidity and mortality.

The Wee1 kinase inhibitor MK1775 suppresses cell growth, attenuates stemness and synergises with bortezomib in multiple myeloma.

Multiple myeloma stem-like cells (MMSCs) are responsible for initiation and relapse, though novel treatment paradigms that effectively eradicate MMSCs are yet to be developed. Selective inhibition of the cell cycle regulatory kinase Wee1 by MK1775 is being explored as a potential anti-cancer therapeutic. We report that higher expression of Wee1 is correlated with poor survival in multiple myeloma (MM). The MM models and patient-derived CD138+ plasma cells are particularly sensitive to the growth-inhibitory effects of the Wee1 inhibitor MK1775. MK1775 induces Mus81-Eme1 endonuclease-mediated DNA damage in S-phase cell cycle that results in a blockade of replication and then apoptosis. Furthermore, MK1775 strongly suppresses the features of stemness in vitro, in vivo and in primary CD138+ cells by decreasing ALDH1+ cell fraction and the expression of ALDH1. In addition, co-treatment of MK1775 with bortezomib is synergistic in vitro and in vivo. Bortezomib, although it enhances ALDH1+ cells, when combined with MK1775 abrogates this stimulatory effect on stemness. Considering MM as an invariably incurable malignancy due to the presence of heterogenic myeloma stem-like cells, our study presents inhibition of Wee1 as a promising targeted therapy for MM and provides a compelling rationale to further investigate the activity of MK1775 against myeloma in clinical settings.

Endomembrane Systems are Reorganized by ORF3a and Membrane (M) of SARS-CoV-2

The endomembrane reticulum (ER) is largely reorganized by SARS-CoV-2 infection, however, detailed molecular mechanisms of ER remodeling by the virus are still not fully understood. Here, we introduce a “plug and playable” proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged ORF3a and membrane (M) proteins of SARS-CoV-2. Through the mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using TurboID, 117 and 191 proteins were robustly determined as interactomes of ORF3a and M, respectively, and many of them overlap with the mitochondrial-associated membrane (MAM) proteome, including RNF5 (E3 ubiquitin ligase). We also found that MAM formation and secreted proteome profiles were largely affected by the expression of ORF3a. Our results imply that SARS-CoV-2 may utilize MAM as a replication site, and we can suggest novel antiviral treatments for the blockade of viral replication in host cells. Funding: This work was supported by the National Research Foundation of Korea (NRF-,2019R1A2C3008463) and the Organelle Network Research Center (NRF-,2017R1A5A1015366). H.W.R. was supported by Creative-Pioneering Researchers Program through Seoul National University. J.Y.M. and M.J. are supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (Information & Communication Technology) (20-BR-01-09). EM data were acquired at Brain Research Core Facilities in KBRI. J.-S.K. are supported by IBS-R008-D1 of the Institute for Basic Science from the Ministry of Science and ICT of Korea. Conflict of Interest: The authors declare no competing interests.

Early antiretroviral therapy limits SIV reservoir establishment to delay or prevent post-treatment viral rebound.

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4-9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years.

Continuous DNA replication is required for late gene transcription and maintenance of replication compartments in gammaherpesviruses.

Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication.

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

Restriction Factors Against Human CMV

Cellular proteins called 'restriction factors' (RFs) form an important component of the innate immune response to viral replication. However, viruses have learned how to antagonize RFs through mechanisms that are specific for each virus. Here, we summarize the general hallmarks of RFs before going on to discuss the specific strategies recruited by some key RFs that strive to hold human CMV (HCMV) infection back, as well as the counter-restriction mechanisms employed by the virus to overcome this innate defense. Such RFs include the cellular constituents of nuclear domain 10 (ND10), and IFI16, a nuclear member of the PYHIN protein family. Viral regulatory proteins, such as IE1 or pp71, try to oppose the ND10-induced blockade of virus replication by either modifying or disrupting this RF. IFI16, on the other hand, inhibits virus DNA synthesis by downregulating the transcription of viral gene UL54; the intruding virus attempts to antagonize IFI16 by mislocalizing it from the nucleus to the cytoplasm via the action of viral protein UL97. Finally, we consider how Viperin, a RF initially thought to inhibit HCMV maturation late during infection, has actually been demonstrated to enhance virus maturation by increasing lipid metabolism and enhancing virus envelopment.

Stable HIV-1 integrase diversity during initial HIV-1 RNA Decay suggests complete blockade of plasma HIV-1 replication by effective raltegravir-containing salvage therapy

BackgroundThere is legitimate concern that minority drug-resistant mutants may be selected during the initial HIV-1 RNA decay phase following antiretroviral therapy initiation, thus undermining efficacy of treatment. The goal of this study was to characterize viral resistance emergence and address viral population evolution during the first phase of viral decay after treatment containing initiation.Findings454 sequencing was used to characterize viral genetic diversity and polymorphism composition of the HIV-1 integrase gene during the first two weeks following initiation of raltegravir-containing HAART in four ART-experienced subjects. No low-prevalence Raltegravir (RAL) drug resistance mutations (DRM) were found at baseline. All patients undergoing treatment received a fully active ART according to GSS values (GSS ≥ 3.5). No emergence of DRM after treatment initiation was detected. Longitudinal analysis showed no evidence of any other polymorphic mutation emergence or variation in viral diversity indexes.ConclusionsThis suggests that fully active salvage antiretroviral therapy including raltegravir achieves a complete blockade of HIV-1 replication in plasma. It is unlikely that raltegravir-resistant HIV-1 may be selected in plasma during the early HIV-1 RNA decay after treatment initiation if the administered therapy is active enough.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells.

To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Induction of Transcription Factor Early Growth Response Protein 1 during HSV-1 Infection Promotes Viral Replication in Corneal Cells

AimsTo understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication.Study DesignRabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection.MethodologyEgr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays.ResultsOur results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus.ConclusionTogether these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

Beta5 integrin is the major contributor to the alphaV integrin-mediated blockade of HIV-1 replication

Beta5 integrin is the major contributor to the alphaV integrin-mediated blockade of HIV-1 replication

β5 Integrin Is the Major Contributor to the αv Integrin-Mediated Blockade of HIV-1 Replication

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. During the differentiation of monocytes to macrophages, adhesion molecules such as integrins are upregulated; therefore, they provide signals that control the process and subsequently may render macrophages more susceptible to HIV-1 infection. Previous work demonstrated that blocking α(v)-containing integrins triggered a signal transduction pathway leading to the inhibition of NF-κB-dependent HIV-1 transcription. In this paper, we show the influence of the different α(v)-coupled β integrins in HIV-1 replication in macrophages. Inhibition of β integrins, either by specific mAbs, small arginine-glycine-aspartic acid (RGD) mimetic compounds, or RNA interference, showed that integrin β(5) was the major contributor to the integrin-mediated blockade of HIV-1 replication. Importantly, such inhibition did not induce changes in cell adhesion to the substrate. In conclusion, our results reveal a significant role of the integrin dimer α(v)β(5) in HIV-1 infection of macrophages.

Amrubicin Targets Nuclear Matrix-Bound Topoisomerase II In Human Myeloid Leukemia Cells

AbstractAbstract 1830Amrubicin, a synthetic anthracycline which is approved in Japan for the treatment of lung cancer, is currently being evaluated in a global phase III trial for second line treatment of SCLC. Anthracyclines can exert cytotoxicity through inhibiting topoiosomerase II (Topo II), resulting in the blockade of DNA transcription and replication. Similar to etoposide, Amrubicin also is thought to induce covalent DNA protein complexes (stabilized DNA-topo II complexes) and double strand DNA breaks. In this study, we compared the cytotoxic and DNA damage-inducing effects of amrubicin to two other Topo II inhibitors and evaluated their effects on purified human Topo II as well as on the MV4-11 leukemia cell line. Cellular cytotoxicity assays demonstrated that amrubicin, etoposide and daunorubicin induced MV4-11 leukemia cell death at EC50's of 0.7 μM, 1.8 μM and 0.2 μM, respectively. In addition, DNA damage as represented by H2AX phosphorylation, was observed following drug treatment but was less extensive with amrubicin than with daunorubicin. One level of DNA organization consists of the folding of chromatin into 50–100 kb loops, which are attached at their bases to the nuclear matrix. The matrix attachment regions (MARS) of DNA contain strong Topo II binding sites and mediate the attachment of the chromatin loops to the nuclear matrix. We examined the effects of amrubicin on purified human Topo II, nuclear matrix-bound Topo II, and Topo II activity in MV4-11 leukemia cells in order to gain insights into the mechanism of amrubicin-induced apoptosis. The results were compared to those obtained with the “classical” Topo II-active drugs, etoposide and daunorubicin. Amrubicin, etoposide and daunorubicin inhibited the DNA strand passage reaction of purified human Topo II by stabilizing the formation of Topo II-DNA cleavable complexes (at IC50 values of 20 μM, 750 μM and 4 mM, respectively). In MV4-11 leukemia cells, cleavable complex stabilization by IC50 concentrations of etoposide and daunorubicin resulted in extensive DNA fragmentation from 1–50 kb. In contrast, DNA cleavage by amrubicin was limited to the formation of >50 kb fragments even after 24 hrs of continuous drug exposure. The >50 kb fragments were similar in size to the 50–100 kb chromatin loops that are attached to the nuclear matrix. The hypothesis that amrubicin inhibits selectively nuclear matrix-bound Topo II resulting in detachment of the chromatin loops from the matrix was examined directly by using isolated nuclear matrix preparations from MV4-11 cells. Amrubicin was much more potent in inducing chromatin loop release from isolated nuclear matrix preparations than either etoposide or daunorubicin despite the fact that all three drugs stabilized Topo II-DNA cleavable complex formation. Chromatin loop release by amrubicin was Mg++ dependent and reversible by 4 mM ATP indicating that it was Topo II mediated. Taken together, our results suggest that in MV4-11 cells amrubicin selectively targets nuclear matrix-bound Topo II, while etoposide and daunorubicin are less selective and induce generalized and extensive DNA damage. Exposure to etoposide and daunorubicin is associated with risk of myelodysplastic syndrome and secondary acute myeloid leukemia, which may be related to their ability to induce generalized DNA damage. As a more selective DNA damaging agent, further clinical development of amrubicin is warranted. Disclosures:Fernandes:celgene corp: Consultancy, Research Funding. Sung:celgene corp: Employment.

Mitochondrial DNA base excision repair and mitochondrial DNA mutation in human hepatic HuH-7 cells exposed to stavudine

The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase γ (Pol γ), resulting in a blockade of mtDNA replication and subsequent disruption of cellular energetics. Because mtDNA Pol γ is not only involved in mtDNA replication but also responsible for mtDNA repair, we hypothesize that mitochondrial oxidative stress leads to changes in the balance between mtDNA repair and mutation following stavudine (d4T) treatment. However, the mechanisms underlying how changes in mtDNA base excision repair (mtBER) lead to mtDNA mutation remain unclear. To test this hypothesis, total mitochondrial repair capability, different steps of mtBER, mtDNA mutations in D-loop, and oxidative stress were all assessed in cultured HuH-7 human hepatoblast cells treated with d4T for 2 weeks. Assessment by denaturing Southern blotting and quantitative PCR revealed that d4T significantly reduced in vivo repair of H 2O 2 damaged mtDNA in HuH-7 cells. d4T reduced total in vitro mtBER and DNA Pol γ capability, but did not affect mtDNA oxoguanine glycosylase and apurinic/apyrimidinic endonuclease activity in HuH-7 cells. In addition, d4T treatment is associated with a significant increase in the frequency of mtDNA mutations in HuH-7 cells. In conclusion, d4T treatment reduces mtBER and contributes mechanistically to NRTI-induced mtDNA mutation. These events may potentially be associated with some diseases linked to mtDNA mutation.

Psoralen-Induced DNA Interstrand Cross-Links Block Transcription and Induce p53 in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner

Psoralen plus UVA light (PUVA) is commonly used to treat psoriasis, a common skin disorder associated with rapid proliferation of cells. PUVA exerts its antiproliferative activity through formation of DNA monoadducts and interstrand cross-links (ICLs). However, this treatment may lead to skin malignancies as a direct result of inducing carcinogenic DNA damage. Inactivation of the p53 tumor suppressor gene is an important event in the development of skin cancer. p53 is rapidly phosphorylated and stabilized in response to DNA damage, and the induction of apoptosis by p53 is an important mechanism by which p53 exerts its tumor-suppressive activity. To better understand the mechanism by which PUVA treatment induces p53, we exposed human skin fibroblasts with PUVA under conditions that differentially produce monoadducts and ICLs and found that psoralen-induced ICLs induced phosphorylation of the Ser-15 site of p53 and apoptosis much more effectively than psoralen-induced monoadducts. The induction of p53 phosphorylation by psoralen ICLs did not require factors believed to be involved in the repair of psoralen ICLs [xeroderma pigmentosum (XP)-A, XP-C, XP-F, Cockayne's syndrome-B, Fanconi anemia] but did require the ataxia-telangiectasia and Rad3-related but not the ataxia-telangiectasia mutated kinase. Psoralen-induced ICLs blocked transcription and replication more efficiently than monoadducts, and induction of p53 and apoptosis correlated with doses causing interference with transcription rather than DNA replication. Our finding that cells underwent apoptosis preferentially during S-phase suggests that the combined blockade of transcription and DNA replication by psoralen ICLs during S-phase elicits a strong apoptotic response.

Contribution of MX dynamin, oligoadenylate synthetase, and protein kinase R to anti-paramyxovirus activity of type 1 interferons in vitro

To determine the contribution of MX dynamin, oligoadenylate synthetase (OAS), and double-stranded RNA-dependent protein kinase R (PKR) to the antiviral effects of type 1 interferons (IFNs) against bovine parainfluenza-3 virus (PI-3V) infection of Vero cells. Vero cell cultures. PI-3V yield was first compared between control and transfected type 1 IFNs-incompetent Vero cells expressing recombinant OAS or MX proteins. Afterwards, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) was used to scale the degree of PKR activation upon infection of Vero cells by PI-3V. Overexpression of OAS did not result in significantly decreased viral replication. Phosphorylated eIF2alpha forms, the hallmark of PKR activation, were not increased in IFNalpha-primed infected Vero cells. Although human MXA contributed to partial blockade of replication of bovine PI-3V, the antiviral effect was not as strong as that of IFNalpha. The powerful anti-Paramyxovirus activity of type 1 IFNs is mediated by noncanonic pathways.

Is the Malononitrilamide FK778 Better for the Prevention of Acute or Chronic Rejection?

Objective The aim of this study was to assess the efficacy of FK778 to prevent acute and chronic allograft rejection compared with other immunosuppressive agents. Materials and Methods Heterotopic Brown-Norway (BN)-to-Lewis rat cardiac transplantations and heterotopic BN-to-Lewis tracheal transplantations were performed to study acute heart rejection and the development of chronic obliterative airway disease (OAD), respectively. Recipients were treated with FK778, tacrolimus, MMF, or sirolimus for 10 days (acute rejection study) or 28 days (chronic OAD study) at varying doses. Results In untreated recipients, cardiac allograft survival was 6.2 ± 0.4 days. FK778 (20 mg/kg), tacrolimus (2 or 8 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly prolonged graft survival to 17.0 ± 2.8, 18.5 ± 2.7, 25.0 ± 2.5, 20.7 ± 3.8, 14.5 ± 2.2, and 23.2 ± 1.5 days, respectively ( P < .05). Tracheal grafts in untreated recipients showed intense infiltration and complete luminal obliteration by day 28. FK778 (20 mg/kg), tacrolimus (1 or 4 mg/kg), MMF (10 or 40 mg/kg), or sirolimus (0.5 or 2 mg/kg) significantly inhibited tracheal luminal obliteration (19.5% ± 16.4%, 44.2% ± 33.6%, 12.3% ± 3.3%, 61.7% ± 18.6%, 18.3% ± 11.3%, 55.0% ± 30.9%, and 8.5% ± 3.5% ( P < .05). All 4 high-dose groups showed similar efficacy. Conclusions When used in therapeutic doses, tacrolimus and sirolimus were more effective than FK778 to prolong cardiac allograft survival. However, with its antiproliferative effects on smooth muscle cells, its good tolerability, and its blockade of cytomegalovirus replication, FK778 proved effective to prevent chronic OAD development. Thus, FK778 may acquire an important role in maintenance therapy for the prevention of long-term fibroproliferative complications.

R5 HIV gp120-mediated cellular contacts induce the death of single CCR5-expressing CD4 T cells by a gp41-dependent mechanism.

The use of CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5) by X4 and R5 human immunodeficiency virus (HIV) envelopes (Env) influences HIV cytopathicity. Here, we have evaluated the role of CCR5 and gp41 in Env-induced cell death occurring during the contacts of uninfected, primary cells with MOLT cells infected with different R5 and X4 HIV isolates. As reported for X4-Env, R5 HIV-infected cells destroyed CD4 T cells expressing the appropriate coreceptor by inducing the formation of syncytia and the death of single target cells. Therefore, only the small (<10%) CCR5+ subset of primary CD4 T cells was sensitive to cellular presentation of R5-Env, and CCR5-CD4 T cells showed complete resistance to R5-Env-mediated cell death. X4- and R5-infected cells killed single primary cells by a common mechanism that was dependent on gp41 function and induced a rapid loss of mitochondrial membrane potential and plasma membrane integrity in target cells. Single-cell death was not affected by the blockade of HIV replication in target cells or G-protein signaling through CXCR4/CCR5. In contrast, caspase inhibition (Z-Val-Ala-Asp-fluoromethylketone) profoundly changed the outcome of cell-to-cell contacts by reducing the number of single dead CD4 T cells and increasing the rate of syncytium formation. In conclusion, X4 and R5 HIV Env share a common gp41-dependent mechanism to kill CD4 T cells during cellular contacts. Env tropism and coreceptor expression but not differential killing mechanisms seem to govern the extent of cytopathic effects induced by HIV infection.

Azathioprine: old drug, new actions.

A knowledge of the biochemical loci of action of 6-MP in the inhibition of nucleic acid synthesis is not sufficient to explain the effects of the thiopurines on the immune system. —Gertrude B. Elion (Winner of 1988 Nobel Prize in Medicine for “important principals of drug development”; codiscovered 6-MP and azathioprine with George Hitchings) (1) Azathioprine is among the oldest pharmacologic immunosuppressive agents in use today. Initially developed as a long-lived prodrug of 6-mercaptopurine (6-MP), it was quickly found to have a more favorable therapeutic index. It was soon found that 6-MP could produce remissions in childhood acute leukemia (1), and later, that azathioprine could prolong renal allograft survival (2). Over the past 50 years, azathioprine has been used in the treatment of hematologic malignancies, rheumatologic diseases, solid organ transplantation, and inflammatory bowel disease. The drug is a purine analog, and the accepted mechanism of action is at the level of DNA (1, 3). Both in vitro and in vivo, azathioprine is metabolized to 6-MP through reduction by glutathione and other sulphydryl-containing compounds and then enzymatically converted into 6-thiouric acid, 6-methyl-MP, and 6-thioguanine (6-TG) (1, 3). Ultimately, azathioprine can then become incorporated into replicating DNA and can also block the de novo pathway of purine synthesis. It is this action that is thought to contribute to its relative specificity to lymphocytes due to their lack of a salvage pathway. However, the effects on the blockade of DNA replication have never fully explained all of the laboratory and clinical findings of azathioprine-induced immunosuppression.