The role of multiparametric ultrasound in the detection of clinically significant prostate cancer.

PURPOSE: Transrectal ultrasound (US) imaging is paramount to the successful completion of prostate biopsies. Certain US features have been associated with prostate cancer (PCa), but their utility remains controversial. We explored the role of multiparametric US (mpUS) in the detection of clinically significant PCa. METHODS: We performed a retrospective cohort study to contrast the findings of prostate MRI and mpUS. Patients who underwent MRI, US and biopsy between 2015 and 2021 were included. Biopsies were performed using a systematic approach (12 cores), as well as with MRI (4 cores/lesion) and US (1 core/lesion) targeting. The US features analyzed consisted of: calcifications, hypoechoic lesions and power or color Doppler positivity. Gleason 3 + 4 or higher was used as to define true positives. Measures of diagnostic accuracy were calculated for the different imaging modalities. RESULTS: The final cohort included 74 patients, of which 24 (32.4%) had clinically significant PCa. The concordance between MRI and US was 63.5%. Seven individuals with discordant results had clinically significant PCa. MRI alone was more sensitive (87.5% vs 75%) but less specific (28% vs 32%) than US alone. An all-inclusive approach considering any suspicious US or MRI finding had a sensitivity of 95.8%. A more restrictive approach, targeting lesions noted in both US and MRI, yielded the highest specificity (50.0%) and accuracy (55.4%). CONCLUSION: Biopsy targeting based on US findings can provide additional diagnostic information that may increase sensitivity or specificity. Additional research into this topic could open the door to a more personalized approach to prostate biopsy.

Patient and public involvement in the design and conduct of a large, pragmatic observational trial to investigate recurrent, high-risk non-muscle-invasive bladder cancer.

BACKGROUND: Patient-centered approaches to research design are particularly important for diseases with complex treatment decision-making, such as recurrent, high-grade non-muscle-invasive bladder cancer (NMIBC). The objective of this article is to describe patient and public involvement (PPI) in designing a large, pragmatic observational trial and to articulate barriers, challenges, and lessons learned for future design. METHODS: Through multistakeholder involvement, a large, pragmatic observational trial was designed to investigate the outcomes of high-risk, recurrent NMIBC, and it was titled Comparison of Intravesical Therapy and Surgery as Treatment Options for Bladder Cancer (CISTO). CISTO's design used the Guidance for Reporting Involvement of Patients and the Public 2 reporting checklist for PPI and built on prior engagement infrastructure in partnership with the Bladder Cancer Advocacy Network. RESULTS: CISTO's PPI began with research prioritization to identify the highest priority questions facing patients with NMIBC. A pragmatic observational study design was selected and refined through stakeholder input. PPI included patients and caregivers organized into an advocate advisory board and clinicians, researchers, payers, and industry representatives organized into an external advisory board. An engagement plan was created to define the stages of PPI and the level and nature of the involvement of each group. PPI was measured quantitatively and qualitatively through evaluation surveys and iterative feedback from board members, with changes made for continuous improvement. CONCLUSIONS: Through intentional PPI, CISTO aims to produce pragmatic and generalizable results that will allow patients to make informed decisions for recurrent, high-risk NMIBC based on their personal experiences. LAY SUMMARY: Involving patients and other stakeholders in research ensures that it reflects the outcomes that matter most to them. This is especially important when research focuses on conditions in which patients face difficult decisions about treatment options. This article describes the key role that stakeholders played in shaping the Comparison of Intravesical Therapy and Surgery as Treatment Options for Bladder Cancer (CISTO) study. It compares treatments for recurrent noninvasive bladder cancer and describes how stakeholders were engaged to design and develop the study and the practices that supported their involvement.

Prospective quality of life in men choosing open vs. robotic radical prostatectomy: long-term results from a racially diverse multi-institutional database.

PURPOSE: To compare 5-year health-related quality of life (HRQoL) outcomes between prostate cancer (CaP) patients who underwent robotic-assisted laparoscopic radical prostatectomy (RALP) versus open radical retropubic prostatectomy (RRP) and assess for racial disparities between Caucasian American (CA) and African American (AA) men undergoing surgery. METHODS: A prospective cohort study of HRQoL data was conducted on patients diagnosed with CaP from 2007 to 2017 and enrolled in the Center for Prostate Disease Research (CPDR) Multicenter National Database. Using the EPIC and SF-36 instruments, changes in urinary, sexual, bowel, and hormonal domains, as well as physical and mental component summary scores were compared across surgery type (RALP versus RRP) at pre-treatment ("baseline"), and annually for 5 years. We further compared HRQoL outcomes in CA and AA men undergoing surgery. Longitudinal HRQoL patterns were modeled using generalized estimating equations (GEE), adjusting for baseline HRQoL and other characteristics. RESULTS: 448 CaP patients (22% AA) met study inclusion criteria, 66% underwent RALP and 34% underwent RRP. At baseline, HRQoL domains were comparable across treatment group (RALP vs. RRP). In the adjusted low-risk cohort, there were only three time points that met a statistically significant HRQoL difference in EPIC scores between RALP and RRP. Urinary function score during year 4 of follow-up showed a 7.5 (95% CI 3.1-11.9, P = 0.01) points difference in favor of RRP. Bowel bother scores favored RRP in year 1 with a difference of 3.1 (95% CI 0.7-5.4, P = 0.04) points, and in year 5 with a difference of 3.8 (95% CI 1.1-6.4, P = 0.03) points. In the intermediate/high-risk cohort, there were no statistically significant differences in any of the domain scores between RALP and RRP during follow-up. CONCLUSIONS: The robotic and open approach to radical prostatectomy led to comparable HRQoL outcomes at a follow-up length of 60 months. No HRQoL racial disparities were found between AA and CA men during long-term follow-up.

Use of Neoadjuvant Chemotherapy in Elderly Patients With Muscle-Invasive Bladder Cancer: A Population-Based Study, 2006-2017.

Objectives/Introduction Neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC) is associated with survival benefit across patients of all ages, yet it is not offered to many elderly patients. We aimed to evaluate age-based disparity in treatment and outcomes of MIBC. Methods Using the National Cancer Database, we identified patients with MIBC from 2006 to 2017. Use of treatment modalities was compared between age groups. A second analysis compared perioperative outcomes and overall survival (OS) in elderly patients (70 years or older) undergoing RC with NAC vs no NAC. Propensity score weighting (PSW) was used for each analysis. Results In 70,911 patients, use of NAC with RC was lower in patients 70 years or older (7.2% vs 20.9%; P < .001). In patients 70 years or older undergoing RC, NAC was associated with shorter inpatient stay (8.5 vs 9.6 days; P < .001), decreased 30-day readmission (8.6% vs 10.6%; P <.001), and lower 30- and 90-day mortality (1.5% vs 3.1%; P = .01; and 4.9% vs 7.7%; P = .003, respectively). On weighted multivariate regressions, NAC predicted shorter length of stay and lower 30-and 90-day mortality. Elderly patients receiving NAC had improved OS compared with RC alone (P = .0011, 2010-2013; P < .001, 2014-2016). Conclusions Despite increased omission of NAC in patients 70 years or older, elderly patients receiving NAC and RC had improved perioperative outcomes and OS compared with those undergoing RC alone. There may be selection bias unaccounted for with our PSW; however, our results provide compelling evidence that NAC does not compromise surgical outcomes in appropriately selected elderly patients. Patients of advanced age who are candidates for RC should be offered NAC.

Does advancement in stapling technology with triple-row and enhanced staple configurations confer additional safety? A matched comparison of 340 stapled ileocolic anastomoses.

BACKGROUND: Over the past few decades, studies have focused on the safety of stapled anastomosis, especially when compared to that of the handsewn technique. However, studies on the improvement of stapling technology are limited. This study aimed to investigate whether linear triple-row staples (tri-staples) had any advantage over double-row staples. METHODS: This is a retrospective review of all cases of functional end-to-end anastomoses with linear staplers performed at two centers between 2005 and 2015. Data were retrieved from a prospectively maintained database. Cases of anastomoses performed with double-row (DS) and triple-row (TS) staples were matched according to propensity scores. The rates of anastomotic leakage, bleeding, reoperation, and 30-day mortality were compared. RESULTS: Functional end-to-end ileocolic anastomoses were performed in 563 consecutive patients during the study period. Double- and triple-row stapling devices were used in 389 and 174 anastomoses, respectively. With propensity score matching, 170 cases were chosen from each group. Both groups showed comparable baseline characteristics. The anastomotic leakage, anastomotic bleeding, and intra-abdominal collection rates were 2.4 and 0% (p = 0.123), 1.2 and 0% (p = 0.499), and 3.5 and 1.2% (p = 0.283) for DS and TS, respectively. The reoperation and 30-day mortality rates were 5.9 and 1.8% (p = 0.048) and 0.6 and 1.2% (p = 1.000) for DS and TS, respectively. The median lengths of stay were 5 and 6 days (p = 0.072) for DS and TS, respectively. CONCLUSION: Anastomoses with triple-row staples tended to have a lower morbidity rate, but a significant advantage over double-row staples was not demonstrated in this study.

Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model.

Despite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV(+) patients. In 2009, an HIV(+) patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5(-/-) donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4(+) T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4(+) T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4(+) T cells, an increase in nonmodified CD4(+) T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.

Simian immunodeficiency virus-induced alterations in monocyte production of tumor necrosis factor alpha contribute to reduced immune activation in sooty mangabeys.

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by persistent viral replication in the context of CD4(+) T cell depletion and elevated immune activation associated with disease progression. In contrast, simian immunodeficiency virus (SIV) infection of African-origin sooty mangabeys (SM) generally does not result in simian AIDS despite high viral loads and therefore affords a unique model in which to study the immunologic contributions to a nonpathogenic lentiviral disease outcome. A key feature of these natural SIV infections is the maintenance of low levels of immune activation during chronic infection. Our goal was to delineate the contribution of monocytes to maintaining low levels of immune activation in SIV-infected SM. Utilizing an ex vivo whole-blood assay, proinflammatory cytokine production was quantified in monocytes in response to multiple Toll-like receptor (TLR) ligands and a specific, significant reduction in the tumor necrosis factor alpha (TNF-α) response to lipopolysaccharide (LPS) was observed in SIV-infected SM. In contrast, monocytes from hosts of pathogenic infections (HIV-infected humans and SIV-infected Asian macaques) maintained a robust TNF-α response. In SIV-infected SM, monocyte TNF-α responses to low levels of LPS could be augmented by the presence of plasma from uninfected control animals. The impact of LPS-induced TNF-α production on immune activation was demonstrated in vitro, as TNF-α blocking antibodies inhibited downstream CD8(+) T cell activation in a dose-dependent manner. These data demonstrate an association between nonpathogenic SIV infection of SM and a reduced monocyte TNF-α response to LPS, and they identify a role for monocytes in contributing to the suppressed chronic immune activation observed in these natural hosts.

The effect of race on treatment patterns and subsequent health-related quality of life outcomes in men undergoing treatment for localized prostate cancer.

INTRODUCTION: Racial differences in Health-Related Quality of Life (HRQoL) after treatment of prostate cancer (PCa) are not well studied. We compared treatment patterns and HRQoL in African American (AA) and non-AA men undergoing active surveillance (AS), radical prostatectomy (RP), or radiation (XRT). METHODS: Men diagnosed with PCa from 2007-2017 in the Center for Prostate Disease Research Database were identified. HRQoL was evaluated using Expanded PCa Index Composite and SF-36 Health Survey. RESULTS: In 1006 men with localized PCa, 223 (22.2%) were AA (mean follow up 5.2 yrs). AA men with low-risk disease were less likely to undergo AS (28.5 vs. 38.8%) and more likely to undergo XRT (22.3 vs. 10.6%) than non-AA men, p < 0.001. In intermediate-risk disease, AA received more XRT (43.0 vs. 26.9%) and less RP (50.5 vs 66.8%), p = 0.016. In all men, RP resulted in worse urinary function and sexual HRQoL compared to AS and XRT. Bowel HRQoL did not vary by treatment in AA men, however, in non-AA men, XRT resulted in worse bowel scores than AS and RP. HRQoL was then compared for each treatment modality. AA men had worse sexual bother (p = 0.024) after RP than non-AA men, No racial differences were found in urinary, bowel, hormonal, or SF-36 scores for men undergoing AS, RP or XRT. CONCLUSION: AA men are less often treated with AS for low-risk disease and are more likely to undergo XRT. AA men experience worse sexual bother after RP, however, the effect of XRT on bowel symptoms is worse in non-AA men.

Patient reported outcomes and health related quality of life in localized prostate cancer: A review of current evidence.

BACKGROUND: Oncological outcomes after localized prostate cancer (CaP) treatment are excellent and generally considered equivalent across treatment modalities. Thus, short, and long-term patient health related quality of life (HRQoL) is an important factor in treatment discussions. The purpose of this review was to assess the impact of treatment modality for localized CaP on HRQoL as reflected by recent published trials. METHODS: We conducted a literature review using the PubMed database for studies published between January 2010 and January 2021. We included randomized control trials and observational cohort studies examining HRQoL in patients with localized CaP treated with active surveillance, radical prostatectomy, external beam radiotherapy or brachytherapy. RESULTS: Four randomized control trials and 15 prospective cohort studies were reviewed. Current evidence suggests that surgery has the largest short and long-term negative effect on sexual function and incontinence but advantages with regards to bowel function and irritative-obstructive urinary symptoms. Radiation therapy mainly impacts urinary irritative symptoms and bowel bother. Short-term HRQoL outcomes for active surveillance are most favorable, however, during long-term follow up, there is no significant difference in comparison to radiation. Long-term global quality of life impact regarding anxiety, mental, emotional well-being, and fatigue seem to be equivalent between treatment modalities. CONCLUSIONS: The choice of primary treatment modality for localized CaP results in a unique impact profile on cancer specific HRQoL in both the short and long-term periods. Understanding the different adverse events profiles can provide a basis for informing patients and clinicians regarding the impact of disease and treatments on quality of life and allow for a better patient centered discussion.

Impact of Age and Race on Health-Related Quality of Life Outcomes in Patients Undergoing Radical Prostatectomy for Localized Prostate Cancer.

OBJECTIVES: To investigate impact of age and race on health-related quality of life (HRQoL) in men undergoing radical prostatectomy (RP) using a prospectively maintained, racially diverse cohort. METHODS: The Center for Prostate Disease Research Multicenter National Database was used to identify patients receiving RP from 2007-2017. The Expanded PCa Index Composite and 36 Item Short-Form Health Survey were completed at baseline and regular intervals. Groups were stratified based on age: <60, 60-70, >70. Longitudinal patterns in HRQoL were assessed using linear regression models, adjusting for baseline HRQoL, demographics, and clinical characteristics. RESULTS: In 626 patients undergoing RP, 278 (44.4%) were <60, 291 (46.5%) were 60-70, 57 (9.1%) were >70. Older men had worse baseline urinary bother (P<.01) and sexual HRQoL (P<.01). Baseline urinary function was similar for older and younger men. Post-RP urinary and sexual HRQoL was significantly lower in men >70. However, when adjusting for baseline HRQoL, race, NCCN risk, and comorbidities, no difference was found between age groups in urinary function or bother, or sexual function. Sexual bother was worse in older men until 48 months post-operatively but subsequently improved to levels similar to younger patients. Race independently affected HRQoL outcomes with older African American men reporting worse urinary function and sexual bother. CONCLUSIONS: When accounting for baseline HRQoL, age does not independently predict worse HRQoL outcomes. Older and younger men experience similar declines in urinary and sexual domain scores after RP. Our findings may be used to better inform patients regarding their expected post RP HRQoL and guide treatment decision-making.

Advances in technology and techniques for transcatheter aortic valve replacement with concomitant peripheral arterial disease.

Aortic stenosis (AS) is a prevalent disease affecting 3.7% of the adult population aged 65 or above. In the past, surgical aortic valve replacement (SAVR) was the only definitive therapy available for the treatment of severe AS. Owing to the invasive nature of open-heart surgery, patients with advanced age and frailty could not benefit from SAVR. The advent of transcatheter aortic valve replacement (TAVR) in the past decade has offered an alternative treatment option for patients with severe AS, particularly those who are deemed to have high surgical risks. Nevertheless, a large proportion of patients also have concomitant peripheral arterial disease (PAD), which increases the risk of peri-procedural vascular complication, and precludes the possibility of transfemoral TAVR owing to inadequate luminal size for delivery system deployment. In this review, the prevalence and outcome of TAVR patients with PAD will be discussed. Furthermore, novel technologies and techniques that enable TAVR to be safely performed using transfemoral or alternative access in patients with severe PAD will be reviewed.

Pathogenic infection of Macaca nemestrina with a CCR5-tropic subtype-C simian-human immunodeficiency virus.

BACKGROUND: Although pig-tailed macaques (Macaca nemestrina) have been used in AIDS research for years, less is known about the early immunopathogenic events in this species, as compared to rhesus macaques (Macaca mulatta). Similarly, the events in early infection are well-characterized for simian immunodeficiency viruses (SIV), but less so for chimeric simian-human immunodeficiency viruses (SHIV), although the latter have been widely used in HIV vaccine studies. Here, we report the consequences of intrarectal infection with a CCR5-tropic clade C SHIV-1157ipd3N4 in pig-tailed macaques. RESULTS: Plasma and cell-associated virus was detectable in peripheral blood and intestinal tissues of all four pig-tailed macaques following intrarectal inoculation with SHIV-1157ipd3N4. We also observed a rapid and irreversible loss of CD4+ T cells at multiple mucosal sites, resulting in a marked decrease of CD4:CD8 T cell ratios 0.5-4 weeks after inoculation. This depletion targeted subsets of CD4+ T cells expressing the CCR5 coreceptor and having a CD28-CD95+ effector memory phenotype, consistent with the R5-tropism of SHIV-1157ipd3N4. All three animals that were studied beyond the acute phase seroconverted as early as week 4, with two developing cross-clade neutralizing antibody responses by week 24. These two animals also demonstrated persistent plasma viremia for >48 weeks. One of these animals developed AIDS, as shown by peripheral blood CD4+ T-cell depletion starting at 20 weeks post inoculation. CONCLUSION: These findings indicate that SHIV-1157ipd3N4-induced pathogenesis in pig-tailed macaques followed a similar course as SIV-infected rhesus macaques. Thus, R5 SHIV-C-infection of pig-tailed macaques could provide a useful and relevant model for AIDS vaccine and pathogenesis research.

Functional polymorphisms of the CCL2 and MBL genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection.

OBJECTIVES: To assess associations between the functional polymorphisms G-2518A at the chemokine (C-C motif) ligand 2 gene (CCL2) and mannose binding lectin (MBL) codon 54 variant (A/B) and susceptibility to SARS. METHODS: We genotyped the CCL2 G-2518A and MBL codon 54 variant (A/B) in 4 case-control populations of Chinese descent, totally consisting of 932 patients with SARS and 982 control subjects. RESULTS: Both the high-CCL2-producing GG genotype and the low-MBL-producing B allele were consistently associated with increased risks of SARS-CoV infection in all 4 case-control populations (joint P = 1.6 × 10(-4) and 4.9 × 10(-8), for CCL2 and MBL respectively), with no interaction between polymorphisms could be detected. Furthermore, all the 4 case-control studies demonstrated a cumulative effect on risk of SARS-CoV infection for the combination of polymorphisms (joint P = 1.3 × 10(-10)). However, tests using the area under the curve (AUC) indicated that at this stage, the polymorphisms were unlikely to be appropriate for risk prediction testing because of low AUC values (all <66%). Additionally, no association was observed between the polymorphisms and severity of SARS. CONCLUSIONS: The CCL2 G-2518A and MBL codon 54 variant have a significantly cumulative effect on increased risk of SARS-CoV infection.

Expression of the memory marker CD45RO on helper T cells in macaques.

BACKGROUND: In humans it has been reported that a major site of the latent reservoir of HIV is within CD4+ T cells expressing the memory marker CD45RO, defined by the mAb UCHL1. There are conflicting reports regarding the expression of this antigen in macaques, the most relevant animal species for studying HIV pathogenesis and testing new therapies. There is now a major effort to eradicate HIV reservoirs and cure the infection. One approach is to eliminate subsets of cells housing the latent reservoir, using UCHL1 to target these cells. So that such studies may be performed in macaques, it is essential to determine expression of CD45RO. METHODS: We have used immunofluorescence and flow cytometry to study cell surface expression of CD45RO on lymphocytes from PBMC, lymphoid, and GI organs of rhesus, pigtailed, and cynomolgus macaques. Both direct and indirect immunofluorescence experiments were performed. FINDINGS: CD45RO is expressed on a subset of CD4+ lymphocytes of all pigtailed, a fraction of rhesus, and neither of the cynomolgus macaques studied. The binding of UCHL1 to macaque cells was of lower avidity than to human cells. This could be overcome by forming UCHL1 multimers. Directly conjugating fluors to UCHL1 can inhibit UCHL1 binding to macaque cells. Patterns of UCHL1 expression differ somewhat in macaques and humans, and from that of other memory markers often used in macaques. CONCLUSIONS: CD45RO, defined with mAb UCHL1, is well expressed on CD4+ cells in pigtailed macaques. Using tissues recovered from latently infected pigtailed macaques we are determining whether UCHL1, or other memory markers, can define the cellular locus of the reservoir. The low avidity of this interaction could limit the utility of UCHL1, in its conventional form, to eliminate cells in vivo and test this approach in macaque models of HIV infection.

Dynamics of envelope evolution in clade C SHIV-infected pig-tailed macaques during disease progression analyzed by ultra-deep pyrosequencing.

Understanding the evolution of the human immunodeficiency virus type 1 (HIV-1) envelope during disease progression can provide tremendous insights for vaccine development, and simian-human immunodeficiency virus (SHIV) infection of non-human primate provides an ideal platform for such studies. A newly developed clade C SHIV, SHIV-1157ipd3N4, which was able to infect rhesus macaques, closely resembled primary HIV-1 in transmission and pathogenesis, was used to infect several pig-tailed macaques. One of the infected animals subsequently progressed to AIDS, whereas one remained a non-progressor. The viral envelope evolution in the infected animals during disease progression was analyzed by a bioinformatics approach using ultra-deep pyrosequencing. Our results showed substantial envelope variations emerging in the progressor animal after the onset of AIDS. These envelope variations impacted the length of the variable loops and charges of different envelope regions. Additionally, multiple mutations were located at the CD4 and CCR5 binding sites, potentially affecting receptor binding affinity, viral fitness and they might be selected at late stages of disease. More importantly, these envelope mutations are not random since they had repeatedly been observed in a rhesus macaque and a human infant infected by either SHIV or HIV-1, respectively, carrying the parental envelope of the infectious molecular clone SHIV-1157ipd3N4. Moreover, similar mutations were also observed from other studies on different clades of envelopes regardless of the host species. These recurring mutations in different envelopes suggest that there may be a common evolutionary pattern and selection pathway for the HIV-1 envelope during disease progression.

Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame.

The biological significance of protective CD8 T-cell-mediated responses against non-traditional alternative reading frame epitopes remains relatively unknown. Cytolytic CD8 T cells (CTL) specific for a non-traditional cryptic MHC class I epitope, SYNTGRFPPL, are critically involved in the protection of mice during infection with the LP-BM5 murine retrovirus. The goal of this study was to determine the functional properties of the protective SYNTGRFPPL-specific CTL during LP-BM5 infection of susceptible BALB/c CD8(-/-) mice. Direct infection experiments and adoptive transfer of CD8 T cells derived from perforin (pfp)(-/-), IFN gamma(-/-), FasL(-/-) and, as a positive control, wild-type BALB/c mice, were utilized to assess the effector mechanisms responsible for protection. Our results indicate that SYNTGRFPPL-specific effector CTL preferentially utilize perforin-mediated cytolysis to provide protection against LP-BM5-induced pathogenesis, whereas CTL production of IFN gamma is not required. Our results also suggest a minimal contribution of FasL/Fas-mediated lysis during the effector response. Collectively, these results provide insight into effector mechanisms utilized by protective CTL directed against non-traditional cryptic epitopes during disease protection.

The kinetics of p53 activation versus cyclin E accumulation underlies the relationship between the spindle-assembly checkpoint and the postmitotic checkpoint.

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21(CIP1/WAF1) were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21(CIP1/WAF1), is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21(CIP1/WAF1). These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.

Alternative translational products and cryptic T cell epitopes: expecting the unexpected.

Although CD8 T cell epitopes have been studied extensively, often overlooked are unconventional cryptic epitopes generated from nontraditional sources of peptides/proteins and/or mechanisms of translation. In this review, we discuss alternative reading frame epitopes, both mechanistically and also in terms of their physiologic importance in the induction of antiviral and antitumor CTL responses. Issues of the influence of cryptic translational products on foreign and self-Ag diversity, thymic selection, and the T cell repertoire; disease pathogenesis; and approaches to vaccine design are discussed in context of the potentially large impact of unconventional epitopes on T cell immunity.

Cytolytic CD8+ T cells directed against a cryptic epitope derived from a retroviral alternative reading frame confer disease protection.

Cytolytic CD8(+) T cells (CTL) are key to the immune response that controls virus infections and mediates disease protection. The ability of CTL to induce apoptosis of infected cells and/or limit viral replication is determined by recognition of processed viral peptide epitopes on the surface of the target cell. An understudied source of MHC class I-presented peptides is the aptly named "cryptic epitopes," defined by their nontraditional methods of generation, including derivation from alternative reading frames (ARFs). Although ARF-encoded epitopes have now been documented in a few systems, their potential functional relevance in vivo has been debated. In this study, we demonstrate the physiological significance of an ARF-derived CTL epitope in a retrovirus-induced disease model. We show that disease-susceptible CD8-deficient mice reconstituted with CTL specific for the retroviral ARF-derived SYNTGRFPPL epitope controlled an infection by the LP-BM5 retrovirus isolate, evidently at the level of viral clearance, resulting in protection of these mice from disease. These data indicate that ARF-derived epitopes are indeed relevant inducers of the immune system and demonstrate the importance of atypically generated peptides as functional Ag with a physiologic role in disease protection.