Recombination-deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution.

Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination-deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination-deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence.

Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging.

Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

Innate Immune Responses in the Neutrophils of Community Dwelling and Nursing Home Elders.

OBJECTIVE: To evaluate innate immune responses of older disabled nursing home residents that may contribute to infectious disease susceptibility, we compared surface markers and signaling efficiency of neutrophils from nursing home residents and community dwelling elders. DESIGN: Observational pilot study. SETTING: Five New Haven, CT area nursing homes and the greater New Haven community. PARTICIPANTS: 15 nursing home residents and 43 community dwelling elders. MEASUREMENTS: Neutrophils were isolated and Toll-like receptor (TLR) and ß2 integrin expression on the surface of unstimulated neutrophils were measured via flow cytometry. Chemokine induction was determined by Quantitative PCR. RESULTS: Surface expression of TLR4 was elevated among nursing home residents compared to community dwellers (mean percent positive cells 33.91 [SE 2.75] vs. 15.67 [SE 1.58], p<0.001), while expression of the ß2 integrins CD11b and CD18 was significantly lower (mean fluorescent intensity 460.8 [SE 49.1] vs. 632.9 [SE 29.5] for CD11b and 59.6 [SE 7.9] vs. 137.6 [SE 4.6] for CD18, p<0.0001). Neutrophils from nursing home residents produced substantially reduced levels of chemokines at baseline and after stimulation. CONCLUSIONS: Because integrins are an important pathway to phagocyte signaling and contribute to adherence and locomotion of neutrophils, reduced ß2 integrin expression may contribute to impaired responses to stimulation and reduced adhesive properties in PMN from nursing home residents. Since integrin CD11b has been shown to negatively regulate TLR4 response, it is plausible that lower levels of CD11b contribute to elevated expression of TLR4.

Plasmacytoid dendritic cells, interferon signaling, and FcγR contribute to pathogenesis and therapeutic response in childhood immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder of childhood characterized by immune-mediated destruction of platelets. The mechanisms underlying the pathogenesis of ITP and the therapeutic efficacy of intravenous immunoglobulins (IVIG) in this disorder remain unclear. We show that monocytes from patients with ITP have a distinct gene expression profile, with increased expression of type I interferon response (IR) genes. Plasma from ITP patients had increased levels of several cytokines indicative of immune activation, including an increase in interferon-α. ITP patients also had an increase in plasmacytoid dendritic cells (pDCs) compared to healthy donors. Therapy-induced remission of ITP was associated with abrogation of the IR gene signature in monocytes without reduction in the levels of circulating interferon-α itself. IVIG altered the ratio of activating/inhibitory Fcγ receptors (FcγRs) in vivo primarily by reducing FcγRIII (CD16). The engagement of activating FcγRs was required for IVIG-mediated abrogation of monocyte response to exogenous interferon-α in culture. Moreover, plasma from ITP patients led to activation of monocytes and myeloid DCs in culture with an increase in T cell stimulatory capacity; this activation depended on the engagement of activating FcγRs and interferon-α receptor (IFNAR) and was inhibited by antibody-mediated blockade of these pathways. These data point to a central role of type I interferon in the pathogenesis of ITP and suggest targeting pDCs and blockade of IR as potential therapeutic approaches in this disorder. They also provide evidence for the capacity of IVIG to extinguish IR in vivo, which may contribute to its effects in autoimmunity.

Central and overlapping role of Cathepsin B and inflammasome adaptor ASC in antigen presenting function of human dendritic cells.

Inflammasomes are increasingly implicated in regulating immunity, but how their activation relates to function of human dendritic cells (DCs) is unknown. Here we show that DC maturation stimuli lead to rapid activation of caspase-1 in human monocyte-derived DCs. RNAi mediated inhibition of the inflammasome component ASC leads to marked inhibition of the capacity of lipopolysachharide (LPS)-matured DCs to stimulate antigen-specific T cells. RNAi mediated inhibition of Cathepsin B (CatB) also similarly inhibits the capacity of human DCs to stimulate immunity. The defective ability of ASC or CatB deficient DCs to stimulate T cells is independent of inflammasome-mediated processing of inflammatory cytokines and also includes DCs loaded with pre-processed peptide. Gene expression profiles of ASC or CatB deficient human DCs show marked overlap with downregulation of genes implicated in DC function. These data demonstrate an important role for ASC and CatB in regulating function of human DCs with overlapping effects on gene expression.

Inhibition of neutrophil function by two tick salivary proteins.

The saliva of hematophagous arthropods contains potent anti-inflammatory and antihemostatic activities that promote acquisition of the blood meal and enhance infection with pathogens. We have shown that polymorphonuclear leukocytes (PMN) treated with the saliva of the tick Ixodes scapularis have reduced expression of beta(2) integrins, impaired PMN adherence, and reduced killing of Borrelia burgdorferi, the causative agent of Lyme disease. Here we describe two Ixodes proteins that are induced upon tick feeding and expressed predominantly in the salivary glands. Using saliva harvested from ticks with reduced levels of ISL 929 and ISL 1373 through targeted RNA interference knockdown, as well as purified recombinant proteins, we show the effects of these proteins on downregulation of PMN integrins and inhibition of the production of O(2)(-) by PMN in vitro. Mice immunized with ISL 929/1373 had increased numbers of PMN at the site of tick attachment and a lower spirochete burden in the skin and joints 21 days after infection compared to control-immunized animals. Our results suggest that ISL 929 and ISL 1373 contribute to the inhibition of PMN functions shown previously with tick saliva and support important roles for these inhibitory proteins in the modulation of PMN function in vivo.

The impact of RNA interference of the subolesin and voraxin genes in male Amblyomma hebraeum (Acari: Ixodidae) on female engorgement and oviposition.

Reducing or replacing the use of chemical pesticides for tick control is a desirable goal. The most promising approach would be to develop vaccines that protect hosts against tick infestation. Antigens suitable for the development of anti-tick vaccines will likely be those essential for vital physiological processes, and in particular those directly involved in feeding and reproduction. In this study genes from Amblyomma hebraeum Koch that encode for subolesin and voraxin were studied in male ticks by RNA interference (RNAi). Males (unfed or fed) were injected with dsRNA of (1) subolesin, (2) voraxin, (3) subolesin plus voraxin or (4) injection buffer, after which they were held off-host overnight and then allowed to feed on rabbits together with normal female A. hebraeum. Females that fed together with male ticks injected with subolesin or subolesin + voraxin dsRNA had a higher rate of mortality, weighed substantially less and produced a smaller egg mass than the controls. However, females feeding with males injected with voraxin dsRNA alone were not significantly different from the controls with respect to mortality, engorged weight or fecundity. However, as assessed by semi-quantitative RT-PCR, voraxin was not silenced in this study, the reasons for which remain unknown. The results of this study suggest that A. hebraeum subolesin is worthy of further testing as a candidate tick vaccine antigen.

Identification of two genes essential for sperm development in the male tick Amblyomma hebraeum Koch (Acari: Ixodidae).

In most ticks of the family Ixodidae, gonad maturation and spermatogenesis are stimulated by the taking of a blood meal. Previous work from this laboratory identified 35 genes that are up-regulated by feeding [Weiss, B.L., Stepczynski, J.M., Wong, P., Kaufman, W.R., 2002. Identification and characterization of genes differentially expressed in the testis/vas deferens of the fed male tick, Amblyomma hebraeum. Insect Biochemistry and Molecular Biology 32, 785-793]. The functions of most of these genes remain unknown. We used RNA interference technology to investigate the consequences of blocking the function of 13 of these genes. Attenuation of the expression of two of these in particular, AhT/VD 8 and AhT/VD 10, correlated with deformities in the testis and abnormalities in spermiogenesis. Furthermore, most females fed in the company of these males did not engorge properly and laid many fewer eggs, most of which were infertile.

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori.

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein (egfp) gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part (up to 14 amino acid residues) of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide (mutant 13 aa), the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue(s) in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue(s). Furthermore, the deletion of Arg2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori.

To study the minimal length required for the secretion of recombinant proteins and silk proteins in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain (FibH) of silkworm Bombyx mori was systematically shortened from the C-terminal. Its effect on the secretion of protein was observed using enhanced green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion proteins was examined under fluorescence microscope. FibH SPs with lengths of 20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to 12 a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP. When 16 a.a. of the FibH SP were used, the secretion of fusion protein was normal and the cleavage site was between the Gly-Ser linker and Met, the starting amino acid of EGFP. These findings are applicable for the expression of foreign proteins in silkworm silk gland. The cleavage site of the SP is discussed and compared with the predictive results of the SignalP 3.0 online prediction program.

Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori.

The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209bp region by overlapping deletion studies showed that a 25bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25bp fragment. These results suggest that this 25bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

Loss of posterior silk gland transcription specificity of fibroin light chain promoter due to absence of 41 bp sequence containing possible inhibitor binding sites.

The gene encoding fibroin light chain protein (FibL) is specifically expressed in the posterior silk gland of silkworm and repressed in other tissues. The binding sites of several transcription factors involved in the silk gland transcription specificity of fibl promoter have been recognized, including SGFB, PSGF and BMFA. Here we report the leak expression of the enhanced green fluorescent protein (EGFP) reporter gene in tissues other than the posterior silk gland in vivo when under the control of a shortened fibl promoter with deletion of the 5' terminal 41 bp sequence, which is located at -650 nt to -610 nt upstream of the fibl transcription starting site. Assay of silk gland specificity of the promoters was performed by observation of green fluorescence in tissues of silkworm larvae following inter-haemocoelic injection of recombinant Autographa californica multiple nuclear polyhedrosis virus carrying the EGFP reporter gene controlled by different lengths of fibl promoters. Our results indicated that availability of the binding sites of several known factors, including SGFB, PSGF and BMFA, is not sufficient for intact silk gland transcription specificity of fibl promoter, and there are possible inhibitor binding sites in the 41 bp sequence (-650 nt to -610 nt) upstream of the transcription starting site which may be required to repress the activity of fibl promoter in other tissues.

Productive infection of Autographa californica nucleopolyhedrovirus in silkworm Bombyx mori strain Haoyue due to the absence of a host antiviral factor.

We have reported that several silkworm strains are permissive to intrahemocoelical infection of Autographa californica nucleopolyhedrovirus (AcNPV), contrary to the general belief that AcNPV cannot infect silkworm. In the present study, we address whether the intrahemocoelical infection of AcNPV to the silkworm was an exceptional phenomenon, and the possible genetic basis underlying it. Wilder range test of 31 strains of silkworm Bombyx mori for intrahemocoelical AcNPV infection led to the identification of 14 permissive strains and 17 nonpermissive strains, indicating that the intrahemocoelical infection of AcNPV to the silkworm was not a rare and isolated phenomenon. Productive infection was shown in permissive silkworms, by EGFP fluorescence in various tissues when expression of reporter gene controlled by a very late viral promoter polh. The viral titer in larval hemolymph of permissive silkworms increased and maintained at a higher level hundredfold more than the initial amount of virus, indicating viral replication. A series of genetic cross experiments suggested the existence of only one dominant host anti-AcNPV gene or a set of genetically linked genes, which prevent AcNPV infection in nonpermissive silkworm strain Qingsong and are absent in permissive silkworm strain Haoyue.

p53 protein activates the transcription of human proliferating cell nuclear antigen in response to 4-nitroquinoline N-oxide treatment.

4-nitroquinoline N-oxide (4-NQO) is a potent mutagen and carcinogen. To elucidate the cellular response to 4-NQO, we studied the transcriptional regulation of human proliferating cell nuclear antigen (hPCNA), an essential protein in DNA replication and repair, after 4-NQO treatment. We found that hPCNA promoter was dose-dependently transactivated by 4-NQO under the concentration of 2 microM via a previously reported p53-binding element located from -236 to -217 upstream of the transcription start site. Based on our western blot analysis, the phosphorylation of serine at the 15th residue (Ser15) of p53 was activated by 4-NQO, whereas the level of p53 in the cells did not change much. It was observed that Staurosporine, a Ser/Thr kinase inhibitor, blocked the Ser15 phosphorylation of p53 and the hPCNA promoter response to 4-NQO simultaneously, suggesting that Ser15 phosphorylated p53 was the 4-NQO-responsive hPCNA regulator. The [3H]-thymidine deoxyribose (TdR) incorporation assay and the comet assay showed that DNA repair was triggered when DNA replication was inhibited after the treatment of 4-NQO, and the hPCNA transactivation seemed to contribute to DNA repair. Taken together, our data indicate that after 4-NQO treatment hPCNA is transactivated by Ser15 phosphorylated p53, and participate in DNA repair.

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test.

Electroporation as a methodology to introduce foreign genes into silkworm eggs was systematically analyzed. The foreign gene in both the newly hatched and 3rd instar larva DNA can be detected by PCR. The amount of foreign gene in 3rd instar larva DNA was about 1/1000 of that in newly hatched larva DNA. The ratio of foreign gene entering into silkworm eggs was voltage dependent and showed significant difference between the tested silkworm strains. When the piggyBac transposon system was applied, the effect of nuclear localization signal (NLS) peptide and the in vitro transcribed transposase mRNA on the transposition rate has been measured. Results showed that the in vitro transcribed transposase mRNA facilitated transposition to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. When linearized vectors containing varied length of flanking homologous sequences around a reporter gene were introduced into silkworm eggs by electroporation, the one with 2.6 kb total arm length gave higher G1 positive ratio than that with total arm length of 1.5 kb and 800 bp.