Determinants of Moloney murine leukemia virus Gag-Pol and genomic RNA proportions.

UNLABELLED: The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/Gag-Pol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of Gag-Pol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA. IMPORTANCE: Viruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.

Oncogenic Myc translocations are independent of chromosomal location and orientation of the immunoglobulin heavy chain locus.

Many tumors are characterized by recurrent translocations between a tissue-specific gene and a proto-oncogene. The juxtaposition of the Ig heavy chain gene and Myc in Burkitt's lymphoma and in murine plasmacytoma is a classic example. Regulatory elements within the heavy chain constant region locus are required for Myc translocation and/or deregulation. However, many genes are regulated by cis-acting elements at distances up to 1,000 kb outside the locus. Such putative distal elements have not been examined for the heavy chain locus, particularly in the context of Myc translocations. We demonstrate that a transgene containing the Ig heavy chain constant region locus, inserted into five different chromosomal locations, can undergo translocations involving Myc. Furthermore, these translocations are able to generate plasmacytomas in each transgenic line. We conclude that the heavy chain constant region locus itself includes all of the elements necessary for both the translocation and the deregulation of the proto-oncogene.

The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is <1% the wild-type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

Enhancement of antibody class-switch recombination by the cumulative activity of four separate elements.

Class-switch recombination of Ab isotype is mediated by a recombinational DNA deletion event and must be robustly upregulated during Ag-driven differentiation of B cells. The enhancer region 3' of the Cα gene is important for the upregulation of switch recombination. Using a transgene of the entire H chain C region locus, we demonstrate in this study that it is the four 3' enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation rather than the 24 kb of DNA in between them. Neither allelic exclusion nor transgenic µ expression is reduced by deletion of the four 3' enhancers. We also test deletions of two or three of the 3' enhancers and show that deletion of more 3' enhancers results in a progressive reduction in both switch recombination and germline transcription of all H chain genes. Nevertheless, we find evidence for special roles for some 3' enhancers; different H chain genes are affected by different 3' enhancer deletions. Thus, we find that the dramatic induction of class-switch recombination during Ag-driven differentiation is the result of an interaction among four separated regulatory elements.

Frailty as a Predictor of Surgical Outcomes Following Femoral Hernia Repair.

BACKGROUND: Femoral hernias are associated with significant morbidity and mortality due to risk of strangulation. Frailty has shown to be strongly associated with adverse outcomes. A modified five-factor frailty index (mFI-5) is a simple validated predictor of postoperative complications and mortality within the ACS-NSQIP® database. This study aims to evaluate the impact of frailty and age on 30-day outcomes after femoral hernia repair. METHODS: Patients who underwent femoral hernia repair were queried using the ACS-NSQIP database (2017) and divided into two groups based on frailty score (FS): Frail (FS = 1-5) and Non-frail (FS = 0). We evaluated the association between postoperative outcomes and frailty, age, sex, presentation, ASA class, timing of surgery, and surgical approaches. Univariate analysis followed by a multivariable logistic regression model was performed to evaluate postoperative morbidity. RESULTS: Of a total of 1,295 patients, 540 (42.7%) were in the Frail group. No differences in sex and race proportions were observed between groups. The Frail group had a higher rate of serious morbidity (4.4% vs 1.9%, P < .001), overall morbidity (7.8% vs 3.4%, P < .010), readmission rate (5.4% vs 2.3%, P = .003), and median (IQR) hospital length of stay (1 [0, 4] vs 0 [0, 1] days, P < .001). In multivariable analysis, male sex, presentation with complication, emergency surgery, and FS were associated with increased odds of overall morbidity. All deaths were in the Frail group. CONCLUSION(S): Frailty, male sex, presentation with obstruction/strangulation, and emergency surgery are independent predictors of increased 30-day morbidity. Thirty-day mortality was noted in the Frail group.

The 3' end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four gamma genes.

The switch in immunoglobulin (Ig) heavy chain class is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and class switch recombination we used transgenic mice with a 230-kilobase bacterial artificial chromosome that included a rearranged VDJ gene and the entire heavy chain constant region locus. In addition to several lines with intact transgenes, we identified two lines in which the heavy chain locus transgene lacked Calpha and everything 3' of it, including the regulatory elements HS3a, HS1-2, HS3b, and HS4. B cells from both lines with the truncated transgenes make abundant transgenic (Tg) VDJCmu transcripts and IgM protein. Deletion of the 3' end of the locus results in dramatically reduced expression of both germline transcripts and switched VDJCH transcripts of the gamma3, gamma2b, gamma2a, and epsilon genes. In addition, the transgenes lacking the 3' end of the locus express reduced amounts of gamma1 germline transcripts and 2-3% of the amount of Tg IgG1 in tissue culture compared with intact transgenes. Finally, switch recombination to gamma1 is undetectable in the transgenes lacking the 3' elements, as measured by digestion circularization-polymerase chain reaction or by the expression of VDJCgamma1 transcripts.

High ligation of the saphenofemoral junction in endovenous obliteration of varicose veins.

BACKGROUND: Endovenous radiofrequency (RF) ablation of the greater saphenous vein has become an accepted treatment modality. This study examines if it is necessary to perform high ligation of the saphenous vein to insure success of the procedure. STUDY DESIGN: A retrospective chart analysis was conducted on 219 patients who underwent RF ablation for venous insufficiency. All procedures were performed by 3 board-certified vascular surgeons. One surgeon always ligated the saphenofemoral junction (SFJ), the second never ligated, and the third ligated selectively. Demographic data were collected and analyzed. RESULTS: A total of 77 patients underwent RF ablation with ligation of the SFJ (group I), and 142 patients underwent ablation without ligation (group II). Both groups had similar ablation success rates (P = .0960), 92% (group I) and 84% (group II). CONCLUSION: Saphenofemoral junction ligation is not indicated on a routine basis to achieve success with endovascular ablation of the greater saphenous vein.

Transgenes of the mouse immunoglobulin heavy chain locus, lacking distal elements in the 3' regulatory region, are impaired for class switch recombination.

The immunoglobulin heavy (H) chain class switch is mediated by a deletional recombination event between µ and γ, α, or ε constant region genes. This recombination event is upregulated during immune responses by a regulatory region that lies 3' of the constant region genes. We study switch recombination using a transgene of the entire murine H chain constant region locus. We isolated two lines of mice in which the H chain transgenes were truncated at their 3' ends. The truncation in both transgenic lines results in deletion of the 3'-most enhancer (HS4) and a region with insulator-like structure and activities. Even though both truncated transgenes express the µ H chain gene well, they undergo very low or undetectable switch recombination to transgenic γ and α constant region genes. For both transgenic lines, germline transcription of some H chain constant regions genes is severely impaired. However, the germline transcription of the γ1 and γ2a genes is at wild type levels for the transgenic line with the larger truncation, but at reduced levels for the transgenic line with the smaller truncation. The dramatic reduction in class switch recombination for all H chain genes and the varied reduction in germline transcription for some H chain genes could be caused by (i) insertion site effects or (ii) deletion of enhancer elements for class switch recombination and transcription, or (iii) a combination of both effects.

Switch recombination and somatic hypermutation are controlled by the heavy chain 3' enhancer region.

Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to approximately 1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

Ultrasound surveillance of endovascular aneurysm repair: a safe modality versus computed tomography.

Routine ultrasound surveillance is adequate and safe for monitoring endovascular aneurysm repairs (EVARs). A retrospective chart review including 160 endograft patients was performed from August 2000 to September 2005. All ultrasound examinations (n = 359) were performed by a board-certified vascular surgery group's accredited laboratory. Registered vascular technologists utilized the same equipment consisting of Siemens Antares high-definition ultrasonography with tissue harmonics and color flow Doppler. An identical protocol was followed by each technologist: scan body and both limbs of the endograft and distal iliac vessels, measure anterior-posterior aneurysm sac size, and detect intrasac pulsatility and color flow. Statistical analysis utilized Pearson's correlation coefficient and the paired t-test. Forty-one endoleaks were discovered out of the 359 exams (11.4%). There were type I (7, 17%), type II (26, 63%), and combined type I with type II (8, 20%) endoleaks. Correlation with computed tomography (CT) was obtained in 35 of these cases. CT discovered three endoleaks that were not seen with ultrasound. However, these particular ultrasound exams were inadequate due to additional factors (bowel gas, body habitus, hernia), which prompted CT investigation and, hence, endoleak discovery. Of the 41 endoleaks found on ultrasound, only 14 were seen on CT. Specifically, 26 type II endoleaks were seen with ultrasound versus only nine during CT. Additional factors addressed included comparison between ultrasound and CT of residual aneurysm sac measurements and conditions limiting ultrasound examination. Although criticized in the past, color flow ultrasonography is a safe and effective modality for surveillance of aortic endografts. Utilizing ultrasound to analyze abdominal aortic aneurysm (AAA) sac dimensions and endoleak detection is statistically sound for screening AAA status post-EVAR.

Induced expression of murine gamma2a by CD40 ligation independently of IFN-gamma.

IgG2a, with gamma2a H chains, is important for protection against viruses and other intracellular pathogens. Although a large portion of IgG2a expression is dependent upon IFN-gamma, some germline transcription and switch recombination to the murine gamma2a H chain gene expression are independent of IFN-gamma. We found that agonistic anti-CD40 Abs injected into IFN-gamma-deficient mice induce a > 200-fold increase in the amount of serum Ig2a, while other Ig isotypes are increased by 16-fold or less. In vitro, ligation of CD40 on B cells, without the addition of other B cell activators or cytokines, results in germline transcription and switch recombination that are largely restricted to the gamma2a gene. These results suggest that some immune responses to infectious agents can result in large amounts of IgG2a expression through ligation of CD40, without the expression of IFN-gamma by Th1 or other cells.

Receptors and counterreceptors involved in NK-B cell interactions.

In addition to the well-documented effect of NK cells on B cell differentiation via their ability to secrete IFN-gamma, NK cells can also induce, via direct cell-cell interactions, germline transcripts (Igamma2a) necessary for switch recombination to IgG2a. Analysis of the ligand-receptor pairs that could be involved in this induction revealed that the expression of CD48 on B cells is crucial for the induction. NK cells from mice with targeted deletions of either the CD2 or the CD244 gene, both of which encode ligands for CD48, are compromised in their ability to induce B cell Igamma2a expression. Interestingly, although CD244 can bind to CD48 with a higher affinity, the ability of NK cells from CD244(-/-) mice to stimulate Igamma2a is not as compromised as NK cells from CD2(-/-) mice. Despite the difference between cell surface receptors that are stimulated by NK cells vs those stimulated by the combination of LPS and IFN-gamma, we show in this study that the initiation of gamma2a germline transcription is regulated by similar cis-acting elements located at the 3' end of the IgH locus. However, NK cells cannot induce the final steps of switch recombination resulting in the production of mature mRNA from recombined DNA. Our findings suggest that these different signaling pathways converge on regulatory elements that are common to germline transcription; however, because NK induction does not result in the final steps of switch recombination, some signals initiated by LPS plus IFN-gamma are not induced by NK cells.

Germline transcription and switch recombination of a transgene containing the entire H chain constant region locus: effect of a mutation in a STAT6 binding site in the gamma 1 promoter.

The switch (S) in H chain class is preceded by germline transcription and then mediated by a DNA recombination event. One of the impediments toward understanding the mechanism is the lack of a system in which a recombinant DNA molecule undergoes cytokine-regulated class S recombination. To study class S recombination, we used transgenic mice with a 230-kb bacterial artificial chromosome that included a rearranged VDJ gene and the entire murine H chain constant region locus. We found that both germline transcription and S recombination to the transgenic gamma1 H chain gene were regulated by IL-4 like that of the endogenous genes. In mice with two or more copies of the H chain locus transgene, both germline transcripts and S recombination took place at levels comparable to those from the endogenous loci. We also prepared a version of the transgene with a 4-bp mutation in a STAT6 binding site in the gamma1 promoter region. On the average, this mutation reduced germline transcription by 80%, but did not change the amount of S recombination in vitro. Among both the wild-type and mutant transgenes, we found no significant correlation between the amount of germline transcripts and the amount of S recombination. We infer that the physiologic level of germline transcription of the gamma1 gene is in excess over the amount required for efficient S recombination.