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CD4 + T cells play a critical role in the response to chronic viral infections during the acute phase and in the partial containment of infections once chronic infection is established. As infection persists, the virus-specific CD4 + T cell response begins to shift in phenotype. The predominant change described in both mouse and human studies of chronic viral infection is a decrease in detectable T helper type (Th)1 responses. Some Th1 loss is due to decreased proliferative potential and decreased cytokine production in the setting of chronic antigen exposure. However, recent data suggest that Th1 dysfunction is accompanied by a shift in the differentiation pathway of virus-specific CD4 + T cells, with enrichment for cells with a T follicular helper cell (Tfh) phenotype. A Tfh-like program during chronic infection has now been identified in virus-specific CD8 + T cells as well. In this review, we discuss what is known about CD4 + T cell differentiation in chronic viral infections, with a focus on the emergence of the Tfh program and the implications of this shift with respect to Tfh function and the host–pathogen interaction. [ABSTRACT FROM AUTHOR]

Paternal aging is associated with increased risk of genetic disease transmission to the offspring. The changes associated with aging arise predominantly through formation of single nucleotide variation through DNA replication errors, as well as possibly chronic exposure to environmental toxins and reactive oxygen species exposure. Several age-related reproductive factors are also contributory, including the systemic hormonal milieu, accumulation of environmental toxin exposure, aging germ cells, and accumulation of de novo genetic and genomic abnormalities in germ cells. In this article we review the age-related genetic and genomic changes that occur in the male germ line. [ABSTRACT FROM AUTHOR]

Objective: To present our experience with laparoscopic partial nephrectomy (LPN) for hilar tumors and evaluate intermediate oncologic and renal functional outcomes. Materials and Methods: A retrospective review of LPN cases performed in 488 patients was performed. Hilar lesions were defined as renal cortical tumors in direct physical contact with the renal artery, vein, or both, as identified on preoperative imaging and confirmed intraoperatively. The clinicopathologic parameters, perioperative course, complications, and oncologic and 6-month renal functional outcomes were analyzed. Results: A total of 488 patients underwent LPN, of which 43 were hilar. The mean tumor size for hilar and nonhilar tumors was 3.6 cm and 3.1 cm, respectively. The mean operative time was shorter for hilar as compared with nonhilar tumors (129.1 minutes vs 141.8 minutes). Mean estimated blood loss was greater in LPN for hilar tumors (311.65 mL vs 298.4 mL). There were no statistically significant differences noted in any of the perioperative parameters investigated despite a higher nephrometry complexity score in the hilar group. Change in estimated glomerular filtration rate at 6 months showed a decrease of 10.9 mL/min and 8.8 mL/min for hilar and nonhilar tumors, respectively (P = NS). There was 1 recurrence detected in the hilar group, with a median follow-up of 41.6 months. Conclusion: In the hands of an experienced laparoscopist, LPN can safely be performed for hilar tumors, with preservation of perioperative outcomes and durable renal functional and oncologic outcomes. [ABSTRACT FROM AUTHOR]

Objective: To better elucidate the prevalence of perceived food sensitivity and characterize the sensitivity pattern in patients with clinically diagnosed chronic prostatitis/chronic pelvic pain syndrome. Methods: A total of 286 men meeting the National Institutes of Health criteria for chronic prostatitis were mailed a validated questionnaire designed to detect the effect of foods, beverages, and/or supplements on pelvic pain symptoms. The questionnaire assessed the effect of 176 individual comestibles on each patient's symptoms. The responses were numerically scored on a scale of −2 to +2, and the mean values were generated for each comestible. In addition, the participants were asked to complete the O'Leary-Sant Symptom and Problem Index and Chronic Prostatitis Symptom Index questionnaires. Results: Of the 286 surveys, 95 were returned, yielding a response rate of 33.2%. Of those subjects who responded, 47.4% reported that the consumption of certain comestibles aggravated their symptoms, with the most aggravating being spicy foods, coffee, hot peppers, alcoholic beverages, tea, and chili. In contrast, the comestibles that alleviated the symptoms the most included docusate, pysllium, water, herbal teas, and polycarbophil. Conclusion: Many patients with chronic prostatitis/chronic pelvic pain syndrome have demonstrable food, beverage, and dietary supplement sensitivities. Dietary changes should be considered in the treatment of these patients. [ABSTRACT FROM AUTHOR]

Objective: To assess intraoperative cognitive and motor skills using audio- and video-based analysis. Materials and Methods: The study enrolled 11 surgeons who were categorized into novice (n = 4), intermediate (n = 4), and expert (n = 3) groups. Categorization of skill level was based on years of experience and as determined by the lead expert surgeon. A total of 32 cases were available for analysis, including 5 robotic and 27 laparoscopic renal cases. For each procedure, video and audio components were recorded and sent for grading to 4 blinded judges. The previously validated global rating scale (GRS) and operation-specific rating scale (ORS), as well as a novel cognitive rating scale (CRS), were used to assess performances. Statistical comparisons were performed by analysis of variance. Results: Comparison of the 3 skill levels using analysis of variance showed that each scale was able to differentiate among the levels (P <.05). The mean scores for the before and after audio GRS, ORS, and CRS showed significant difference between the novice, intermediate, and expert groups, demonstrating construct validity. Conclusion: The use of intraoperative audio is an innovative way to assess the cognitive ability of the surgeon. The CRS demonstrated construct validity. The addition of the CRS to the already validated GRS and ORS may serve as a reliable system to objectively evaluate laparoscopic and robotic surgical skill. [ABSTRACT FROM AUTHOR]

Objectives: To prospectively compare a novel type of valveless trocar that creates a curtain of pressurized carbon dioxide [CO2] gas (which maintains pneumoperitoneum at a lower gas flow rate) with standard trocars; to quantify the volume of CO2 used; and to characterize CO2 elimination during laparoscopic renal surgery. Methods: A total of 51 patients undergoing laparoscopic renal surgery by a single surgeon were prospectively evaluated using either the valveless trocar (n = 26) or standard trocars (n = 25). Patient demographics, operative time, volume of CO2 gas consumed, CO2 elimination, perioperative parameters, and postoperative complications were recorded and analyzed. Results: Both patient cohorts were comparable in their preoperative demographics, including body mass index, the number of patients with chronic obstructive pulmonary disease, and smoking history. Mean operative time was lower in the valveless trocar cohort (124.1 minutes) compared with the conventional trocar group (145.6 minutes), P = .047. Use of the valveless trocar was associated with a lower volume of intraoperative CO2 consumed (120.0 ± 82.8 vs 300.6 ± 191.5; P < .001) and reduced CO2 elimination compared with standard trocar use after the first 16 minutes of insufflation (P < .05). Minimal complications occurred, including 2 cases of subcutaneous emphysema in the valveless trocar group, and 1 case of respiratory acidosis in the conventional trocar group. Conclusions: Use of a valveless trocar significantly reduced CO2 consumption during transperitoneal laparoscopy. The valveless trocar also demonstrated significantly reduced CO2 elimination and absorption when compared with the standard trocar. [ABSTRACT FROM AUTHOR]

Objectives: To describe laparoendoscopic single site (LESS) donor nephrectomy procedure through a Pfannenstiel incision. Laparoscopic donor nephrectomy has become the standard approach in harvesting kidneys from live donors. This is usually performed through 3 ports placed in a triangular manner in addition to the Pfannenstiel incision where the kidney is removed. Methods: Through a 5 cm Pfannenstiel incision, three 5 mm ports were placed in a triangular manner. A 5 mm flexible-tip laparoscope was used to perform laparoscopic donor nephrectomy. Before ligating the renal hilum, the superior midline trocar was exchanged for a 12-mm trocar to allow for an Endo-GIA stapler. After the kidney was placed in the entrapment sac, the anterior rectus fascia between the 2 midline ports was incised and the kidney was removed. After closure of the fascial defects, the Pfannenstiel incision was closed in a subcuticular manner. Results: LESS Pfannenstiel donor nephrectomy was successfully performed in 6 patients without standard laparoscopic or open conversion. No additional needlescopic instruments were used. The median age was 46 years with median body mass index of 28.3 kg/m2. The median operative time was 142 minutes with a median warm ischemia time of 5 minutes. Median hospital stay was 2 days and the median pain score at discharge was 0. None of the patients received transfusions perioperatively and none had peri-operative complications. Conclusions: LESS Pfannenstiel donor nephrectomy offers the benefits of improved cosmesis over the standard laparoscopic donor nephrectomy. Prospective randomized trials are needed to compare the postoperative pain levels in between these 2 techniques. [Copyright &y& Elsevier]

Abstract: Permanent prostate brachytherapy (PPB) is an established and successful treatment option for localized prostate cancer. Under ultrasound guidance, the procedure can be performed in the ambulatory setting and represents a conformal, ablative technique. For low-risk patients, PSA control is expected in over 90% with outcomes now reported past 12 years. High-risk patients can benefit from PPB as part of a program that may include external beam therapy and androgen ablation. Morbidity tends to center on urinary function, but with modern adaptive techniques, this can be minimized. Sexual functioning is likely disturbed less with PPB than with surgery or external beam radiation therapy. Finally, cost benefit analysis confirms PPB as the most attractive radiation option. With the incidence of prostate cancer still rising, PPB remains a key treatment option in the successful management of prostate cancers. [Copyright &y& Elsevier]

Recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants Mu and C.1.2 have spike proteins with mutations that may confer resistance to natural and vaccine-elicited antibodies. Analysis of neutralizing antibody titers in the sera of vaccinated individuals without previous history of infection and from convalescent individuals show partial resistance of the viruses. In contrast, sera from individuals with a previous history of SARS-CoV-2 infection who were subsequently vaccinated neutralize variants with titers 4- to 11-fold higher, providing a rationale for vaccination of individuals with previous infection. The heavily mutated C.1.2 spike is the most antibody neutralization-resistant spike to date; however, the avidity of C.1.2 spike protein for angiotensin-converting enzyme 2 (ACE2) is low. This finding suggests that the virus evolved to escape the humoral response but has a decrease in fitness, suggesting that it may cause milder disease or be less transmissible. It may be difficult for the spike protein to evolve to escape neutralizing antibodies while maintaining high affinity for ACE2. [Display omitted] • Mu and C.1.2 are relatively resistant to vaccine-elicited antibodies • Neutralization resistance is caused by R346K, Y449H, E484K, and NTD mutations • Previously infected vaccinated individuals neutralize all variants • The C.1.2 spike is highly resistant but has decreased avidity for ACE2 Tada et al. show that infection with SARS-CoV-2 followed by vaccination results in broadly neutralizing antibody. The C.1.2 variant has a highly mutated spike and is the most neutralization-resistant variant; however, its affinity for ACE2 is decreased. Thus, the virus cannot evolve to escape humoral response without becoming less fit. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin A components, retinol and retinoic acid, are fat-soluble micronutrients and critical for many biological processes, including vision, reproduction, growth, and regulation of cell proliferation and differentiation. The cellular uptake of Vitamin A is through specific interaction of a plasma membrane receptor with serum retinol-binding protein. Human serum albumin (HSA), as a transport protein, is the major target of several micronutrients in vivo. The aim of present study was to examine the interaction of retinol and retinoic acid with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various retinoid contents. FTIR, UV–vis, CD and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant and the effects of complexation on protein secondary structure. Structural analysis showed that retinol and retinoic acid bind non-specifically (H-bonding) via protein polar groups with binding constants of K ret =1.32 (±0.30)×105 M−1 and K retac =3.33 (±0.35)×105 M−1. The protein secondary structure showed no alterations at low retinoid concentrations (0.125mM), whereas at high retinoid content (1mM), an increase of α-helix from 55% (free HSA) to 60% and a decrease of β-sheet from 22% (free HSA) to 18% occurred in the retinoid–HSA complexes. The results point to a partial stabilization of protein secondary structure at high retinoid content. [Copyright &y& Elsevier]

Non-obstructive azoospermia is a distinct diagnosis within male infertility in which no sperm is found in the ejaculate as a result of spermatogenesis failure. Because of the increased prevalence of genetic abnormalities in men with non-obstructive azoospermia, male infertility guidelines recommend screening for karyotype abnormalities and Y chromosome microdeletions in this population. Numerous karyotype abnormalities may be present resulting in impaired spermatogenesis, including: Klinefelter syndrome, translocations, and deletions. Y chromosome microdeletions of the AZFa, AZFb, AZFc subregions all can also result in non-obstructive azoospermia with the possibility of sperm being present if only the AZFc subregion is deleted. While these are the two genetic tests recommended by the guidelines, nearly 50%–80% of non-obstructive azoospermia has no identifiable cause and is deemed idiopathic. Several other genetic defects can lead to non-obstructive azoospermia including Kallmann syndrome, mild androgen insensitivity syndrome, and TEX11. While many additional candidate genes have been proposed, many have yet to be verified or are so infrequent in the population that screening is cost-ineffective. Much research is still required in the genetics of non-obstructive azoospermia and will require multi-institutional initiatives to better understand the genetics of condition. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the specific contribution of ilioinguinal (II) and iliohypogastric (IH) nerve injury and referred pain to interstitial cystitis/bladder pain syndrome and patient-reported chronic pelvic pain, and to enumerate the effects of II and IH nerve resection on the pain and voiding symptoms in patients with IC/BPS.Materials and Methods: This was a prospective cohort study of 8 patients with ICS/BPS who had prior abdominal surgery. All patients received diagnostic image guided T12/L1 nerve blocks, followed by II and IH nerve resections. Validated O'Leary-Sant ICS symptom indices (OSPI) and pelvic pain and urgency/frequency patient symptoms scale (PUF) scores were collected at specified intervals pre- and post-operatively.Results: Median scores at pre-operative (OSPI 13.9, PUF 20.4) and 1 week time points (OSPI 5.9, PUF 11), as well as differences between pre-operative and 10 month time points (OSPI 3.7, PUF 6) were all statistically significant (P = .008 and .009 at 1 week, and .007 and .008 at 10 months, for OSPI and PUF respectively). The mean difference in score from pre-operative to longest follow-up as measured by the OSPI was -14.4 (P < .001) and by PUF -10.3 (P < .001). All time points registered demonstrated improvement in pain scores. There were no surgical complications or adverse events.Conclusion: II and IH nerve resection may be an effective and durable treatment option for those with prior abdominal surgery who have referred interstitial cystitis/bladder pain syndrome pain from these injured nerves. [ABSTRACT FROM AUTHOR]

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56 hi versus CD56 dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis I (MPS I) is due to deficient activity of the 70 KD lysosomal enzyme α-L-iduronidase (IDUA) and results in an inability to degrade glycosaminoglycans in many cells. Severe MPS I results in cardiac disease, skeletal deformities, hearing and vision abnormalities, and mental retardation. We have previously reported that neonatal gene therapy with a high dose of a retroviral vector (RV) expressing canine IDUA resulted in stable expression and marked clinical improvements. However, since most patients with MPS I are not diagnosed at birth, it is necessary to test if gene therapy can be effective in older mice. However, a CTL responseoccurred after gene therapy to adults using the RV designated hAAT-cIDUA-WPRE for which the long terminal repeat (LTR) or the liver-specific human α1-antitrypsin promoter (hAAT) are in the same orientation. Although the liver is the major site of expression with this vector, expression also occurs at early times in spleen cells of an unknown phenotype. We hypothesized that an RV with liver-specific expression might avoid expression in antigen-presenting cells, and thus be unable to induce a CTL response. An RV was developed that contained a cassette with the hAAT promoter, a splice site, the canine IDUA cDNA, and a polyadenylation site in the opposite orientation to the LTR. This vector expressed IDUA activity well in HepG2 (human hepatoma) cells, but not in NIH 3T3 cells. This vector was injected IV into adults after hepatocyte replication was induced with hepatocyte growth factor (HGF). Transduced cells can secrete IDUA into blood, and serum IDUA activity reflects the stability of expression. Although serum IDUA expression was high at 1 week, it fell to low levels at 1 month. These data suggest that liver-restricted expression cannot prevent a CTL response against this lysosomal enzyme. We speculate that APCs might take up enzyme with M6P, and present its peptides. An alternative approach to prolong expression was to inhibit the immune system at the time of transfer of 1×109 transducing units/kg of hAAT-cIDUA-WPRE. CTLA4-Ig is an immunosuppressive agent that binds to CD80 and CD86 and prevents them from activating CD28, anti-CD4 antibody can deplete T cells, and anti-CD40 ligand can block co-stimulation. Although short-term administration of any agent alone did not result in long-term expression, prolonged CTLA4-Ig led to long-term (6.5 month) expression. Alternatively, 2 weeks of CTLA4-Ig combined with 2 days of anti-CD4 or with 2 days of anti-CD40 ligand led to long term expression. Mice that received adult gene transfer with successful immunomodulation had an average of 88+/−11 units (U)/ml of IDUA activity in serum. Bone mineral density and bone radiographs were normal (N=6), the a-wave of an electroretinograms was significantly improved (N=4), and 3 of 4 mice had improved echocardiograms. However, hearing was not improved (N=3). We conclude that adult gene therapy with short term immunomodulation can reduce some manifestations of disease in MPS I mice.Molecular Therapy (2006) 13, S378–S379; doi: 10.1016/j.ymthe.2006.08.1077 [ABSTRACT FROM AUTHOR]

Objective: To examine the long-term efficacy of prostatic urethral lift (PUL) for treating men, with or without an obstructive median lobe (OML), who suffer from lower urinary tract symptoms attributed to benign prostatic hyperplasia.Materials and Methods: A retrospective review was performed on all consecutive PUL cases with or without OML from October 2017 to November 2019 by a single academic surgeon. Outcomes were measured using the International Prostate Symptoms Survey (IPSS) with quality of life (QoL) scores. Comparative testing and mixed-effects linear regression analysis were utilized with significance set at α = 0.05 and performed with Stata (College Station, TX).Results: A total of 110 PUL procedures were performed on 106 patients (4 repeat surgeries) with a median age of 66.5 (interquartile range: 60.4-73.7). Twenty-three patients (21.7%) had an OML. Following PUL, patients in both groups showed significant improvements in IPSS (P <.001) and QoL (P <.001) scores. When stratifying by OML and controlling for confounding, IPSS scores in both groups displayed statistically significant improvement at follow-up visits, with no statistically significant intergroup difference (P = .32). The same held true for QoL improvements, with no statistically significant difference between groups (P = .18). The presence of an OML resulted in minimal effects on perioperative outcomes and complications but required significantly more implants (P = .008).Conclusion: PUL is a minimally invasive procedure effective at reducing lower urinary tract symptoms and improving the lives of men with benign prostatic hyperplasia without incurring risks of serious complications. These improvements were equivalent and upheld in patients who presented with OML. [ABSTRACT FROM AUTHOR]

Abstract: The design and synthesis of novel copolymers that consist of a linear polyoxyethylene block and a dendrimeric poly(ester-sulfide) block with potential applications in drug delivery are described. The dendrimers bear hydroxyl or acrylate functional groups and were prepared via a divergent approach using readily available methoxypoly(ethylene glycol) (mPEG), acryloyl chloride, and 1-thioglycerol as building blocks. All reactions involved occurred efficiently and purification steps consisted of washing the reaction mixture with aqueous solutions and precipitation of the product in diethyl ether. [Copyright &y& Elsevier]

Objective: To better understand patient decision-making and genital satisfaction associated with postorchiectomy testicular prosthesis (TP) implantation in patients with germ cell tumors of the testicle.Materials and Methods: An electronic survey to assess TP decision-making and genital satisfaction was distributed to patients via an institutional database (n = 70) and social media outlets (n = 167). Statistical analyses were performed using chi-square tests for categorical variables, Wilcoxon-Mann-Whitney tests for continuous variables, and multivariate regression analyses to identify independent predictors of receiving a prosthesis, genital satisfaction, and prosthesis satisfaction.Results: 24.9% of respondents elected to receive a TP, but 42% of men without a prosthesis reported never being offered one. Identifying as a heterosexual man (2.86) and receiving a TP (odds ratio = 3.29) were both positive predictors of overall genital satisfaction. Having the orchiectomy performed at an academic institution (odds ratio = 2.87) was a positive predictor of testicular prosthesis TP placement. 89.8% of TP recipients were satisfied with the look of their prosthetic, but only 59.3% of respondents were satisfied with prosthetic feel.Conclusion: There are high levels of genital satisfaction in those who elect to receive a TP postorchiectomy. Associations between TP placement, genital satisfaction, and sexuality merit further investigation. Our results also indicate that patients who pursue an orchiectomy at an academic institution are more likely to receive a TP. The use of social media to recruit study participants in urology should be explored further. [ABSTRACT FROM AUTHOR]

Abstract Opioid pain medications are overprescribed, but few data are available to help in appropriate tailoring of postdischarge opioid prescriptions after surgery. Prior studies are retrospective and based on incomplete responses (<50%) to questionnaires, with small sample sizes for any particular surgery. The ORIOLES initiative was a prospective cohort study (2017–2018) designed to measure postdischarge opioid prescribing and use and clinical predictors of use for consecutive patients after radical prostatectomy. The objectives were to establish a postdischarge opioid reference value to meet the needs of >80% of patients and compare open and robotic surgery. A total of 205 adult patients were enrolled, with 100% completing follow-up. In units of oral morphine equivalents (OMEQ), a median of 225 mg was prescribed and 22.5 mg used. There was no difference by surgical approach or among patients with a history of pain-related diagnoses. Overall, 77% of postdischarge opioid medication was unused, with 84% of patients requiring ≤112.5 mg OMEQ. Only 9% of patients appropriately disposed of leftover medication. Approximately 5% reported continued incisional pain due to surgery at 30 d, but none required continued opioid medication use. Prescribing more opioids was independently associated with greater opioid use in adjusted models. Patient summary In this report, we looked at opioid medication use following discharge after radical prostatectomy. We found that 77% of opioid pain medication prescribed was unused, with 84% of patients using less than half of their prescription. Prescribing more opioids was associated with greater use; only 9% of patients appropriately disposed of leftover medication. Take Home Message In the ORIOLES study, 77% of opioid pain medication prescribed after radical prostatectomy was unused, with 84% of patients using less than half their prescription. Prescribing more opioids was associated with greater use; only 9% of patients appropriately disposed of leftover medication. [ABSTRACT FROM AUTHOR]

Objective: Judicious opioid stewardship would match each patient's prescription to their true medical necessity. However, most prescribing paradigms apply preset quantities and clinical judgment without objective data to predict individual use. We evaluated individual patient and in-hospital parameters as predictors of post-discharge opioid utilization after radical prostatectomy (RP) to provide evidence-based guidance for individualized prescribing.Methods: A prospective cohort of patients who underwent open or robotic RP were followed in the Opioid Reduction Intervention for Open, Laparoscopic, and Endoscopic Surgery (ORIOLES) initiative. Baseline demographics, in-hospital parameters, and inpatient and post-discharge pain medication utilization were tabulated. Opioid medications were converted to oral morphine equivalents (OMEQ). Predictive factors for post-discharge opioid utilization were analyzed by univariable and multivariable linear regression, adjusting for opioid reduction interventions performed in ORIOLES.Results: Of 443 patients, 102 underwent open and 341 underwent robotic RP. The factors most strongly associated with post-discharge opioid utilization included inpatient opioid utilization in the final 12 hours before discharge (+39.6 post-discharge OMEQ if inpatient OMEQ was >15 vs. 0), maximum patient-reported pain score (range 0-10) in the 12 hours before discharge (+27.6 OMEQ for pain score ≥6 vs. ≤1), preoperative opioid use (+76.2 OMEQ), and body mass index (BMI; +1.4 OMEQ per 1 kg/m2). A final predictive calculator to guide post-discharge opioid prescribing was constructed.Conclusions: Following RP, inpatient opioid use, patient-reported pain scores, prior opioid use, and BMI are correlated with post-discharge opioid utilization. These data can help guide individualized opioid prescribing to reduce risks of both overprescribing and underprescribing. [ABSTRACT FROM AUTHOR]

Abstract: Background: Black and premenopausal patients have been shown to have poorer stage for stage survival than the overall population. The purpose of this study was to define the effects of age and race on axillary lymph node involvement at a Midwestern safety-net hospital. The hypothesis was that black patients under the age of 50 would be found to have increased rates of axillary involvement in breast cancer. Methods: A retrospective case review was performed of 184 breast cancer patients from 2000 to 2005. Statistical analysis was performed by race and age. Patients under 50 years of age were defined as premenopausal. Results: The overall rate of axillary involvement was 47.8%. Black patients had an overall rate of axillary involvement of 52.9%. However, premenopausal black patients had a 70.8% rate of axillary involvement (P < .05). Premenopausal white patients had a 46.3% rate of axillary involvement. Logistic regression analysis was performed, and premenopausal age and tumor size were found to be independent predictors of positive lymph node status in black patients. Conclusion: In our study, premenopausal black patients had a much higher rate of axillary lymph node involvement than any other group. This finding was consistent even when tumor size was taken into account. More research needs to be done to better define this difference and to detect this disease at an earlier stage. [Copyright &y& Elsevier]

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient α-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human α1-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 ± 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.Molecular Therapy (2007) 15 8, 1423–1431. doi:10.1038/sj.mt.6300201 [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis I (MPS I) is caused by deficient α-l-iduronidase (IDUA) activity and results in the accumulation of glycosaminoglycans and multisystemic disease. Gene therapy could program cells to secrete mannose 6-phosphate–modified IDUA, and enzyme in blood could be taken up by other cells. Neonatal retroviral vector (RV)–mediated gene therapy has been shown to reduce the manifestations of murine MPS I; however, intravenous injection of RV into adults was ineffective owing to a cytotoxic T lymphocyte (CTL) response against transduced cells. In this study, prolonged inhibition of CD28 signaling with CTLA4-Ig, or transient administration of CTLA4-Ig with an anti-CD40 ligand antibody or with an anti-CD4 antibody, resulted in stable expression in most mice that received RV as adults. Mice with stable expression had 81 ± 41U/ml IDUA activity in serum. This resulted in reductions in bone disease, improvements in hearing and vision, and reductions in biochemical and pathological evidence of lysosomal storage in most organs. Improvements in brain were likely due to diffusion of enzyme from blood. However, aortic disease was refractory to treatment. This demonstrates that most manifestations of MPS I can be prevented using adult gene therapy if an immune response is blocked.Molecular Therapy (2007) 15 5, 889–902. doi:10.1038/mt.sj.6300112 [ABSTRACT FROM AUTHOR]

Although gene therapy has reduced manifestations of genetic diseases, immune responses can abrogate the effect. One approach to inducing tolerance is to perform gene transfer in newborns when the immune system is immature. We demonstrate here that the dose of retroviral vector (RV) is important in mice, as mucopolysaccharidosis I (MPS I) mice that received neonatal intravenous gene therapy with a high dose of a canine α-L-iduronidase (cIDUA)-expressing RV had stable expression, while those that received a low dose did not. It was unclear, however, if neonatal transfer with any dose could induce tolerance in large animals. Therefore, newborn MPS I cats were injected intravenously with the RV expressing cIDUA. Although this resulted in high serum IDUA activity due to secretion by transduced cells, expression fell due to a CTL response. Cats that transiently received the immunosuppressive agent CTLA4-Ig did not develop a CTL response. In contrast, MPS I dogs, which can respond immunologically to canine IDUA, had stable serum IDUA activity after neonatal gene therapy. We conclude that cats, but not dogs, mount a potent CTL response to canine IDUA after neonatal gene therapy, which can be prevented with transient CTLA4-Ig.Molecular Therapy (2006) 14, 5–13; doi: 10.1016/j.ymthe.2006.03.015 [ABSTRACT FROM AUTHOR]

Abstract: Mucopolysaccharidosis I (MPS I) due to deficient α-l-iduronidase (IDUA) activity results in accumulation of glycosaminoglycans in many cells. Gene therapy could program liver to secrete enzyme with mannose 6-phosphate (M6P), and enzyme in blood could be taken up by other cells via the M6P receptor. Newborn MPS I mice were injected with 109 (high dose) or 108 (low dose) transducing units/kg of a retroviral vector (RV) expressing canine IDUA. Most animals achieved stable expression of IDUA in serum at 1240 ± 147 and 110 ± 31 units/ml, respectively. At 8 months, untreated MPS I mice had aortic insufficiency, increased bone mineral density (BMD), and reduced responses to sound and light. In contrast, MPS I mice that received high-dose RV had normal echocardiograms, BMD, auditory-evoked brain-stem responses, and electroretinograms. This is the first report of complete correction of these clinical manifestations in any model of mucopolysaccharidosis. Biochemical and pathologic evaluation confirmed that storage was reduced in these organs. Mice that received low-dose RV and achieved 30 units/ml of serum IDUA activity had no or only partial improvement. We conclude that high-dose neonatal gene therapy with an RV reduces some major clinical manifestations of MPS I in mice, but low dose is less effective. [Copyright &y& Elsevier]

[Display omitted] • New chitosan derivatives containing triethylammonium chloride or triethylphosphonium chloride and their PEGylated analogs have been synthesized and characterized. • The derivatives efficiently bind pDNA at low weight ratio of derivative:pDNA of 2.8:1. • Apoptosis measurements by flow cytometry indicated that the derivatives containing triethylammonium chloride or triethylphosphonium chloride and PEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL. • The present study provides evidence that these PEGylated derivatives are safe and effective vectors for efficient nucleic acid delivery. Among cationic polymers, chitosan (CS) is a promising non-viral vector due to its biocompatibility, biodegradability and safety profile. However, its application for gene delivery is hindered by its poor solubility at physiological pH. In this study, novel water-soluble chitosan derivatives containing triethylphosphonium chloride ((N-(1-carboxybutyl-4-triethylphosphonium chloride) chitosan = TEPB-CS)) or triethylammonium chloride ((N-(1-carboxybutyl-4-triethylammonium chloride) chitosan = TEAB-CS)) with 4-carbon spacers separating the cationic units from the chitosan backbone were synthesized by carbodiimide mediated coupling reactions. Two different degrees of substitution of quaternary phosphonium (43% and 21%) and ammonium (40.6% and 26%) were synthesized. The polymers with higher degrees of substitutions displayed higher solubility in aqueous media and were further grafted with mPEG (TEAB-CS-g-mPEG and TEPB-CS-g-mPEG) and used for DNA complexation, cytotoxicity, apoptosis, and gene transfection studies. Chemical characterization of the above polymers was performed by FTIR, 1H NMR, and 31P NMR, and the degree of substitution of the grafted phosphonium and ammonium and mPEG was calculated by 1H NMR. The polymers efficiently bound pDNA at weight ratio of polymer:pDNA of 2.8:1. From dynamic light scattering experiments, the mean diameters of the calf thymus DNA complexes with the polymers, in water and phosphate buffer (pH 7.4), were in the range of 140 – 330 nm. Zeta potential of these complexes in water became positive as the weight ratios of the polymers/DNA exceeded 2:1. Most of these polymers mildly affect the viability of HeLa cells with a final concentration of 10 µg/mL or higher. Cell viability was only modestly compromised in the presence of 100 µg/mL of TEPB-CS-g-mPEG, and this derivative was also found to display the highest transfection efficiencies among all four polymers. Apoptosis measurements by flow cytometry indicated that both TEAB-CS-g-mPEG and TEPB-CS-g-mPEG induce no perceptible and mild apoptosis with their concentration at 100 µg/mL, respectively. Together, the present study provides evidence that these PEGylated TEPB-CS and TEAB-CS are safe and effective vectors for efficient nucleic acid delivery. [ABSTRACT FROM AUTHOR]

Although gene therapy has reduced manifestations of genetic diseases, immune responses can abrogate the effect. One approach to induce tolerance is to perform gene transfer in newborns when the immune system is immature. This has allowed a therapeutic response to be achieved for lysosomal storage diseases and hemophilia in mice. However, it is unclear if this approach would be effective in large animals with more-mature immune systems at birth, and it is unlikely that this will be effective in humans. Immune responses will likely pose a tremendous hurdle for patients with null mutations, and identification of a large animal model with a sufficiently mature immune system at birth should facilitate preclinical studies to identify ways to block an expected immune response in humans. Gene therapy experiments were performed in mucopolysaccharidosis I (MPS I) cats, which are deficient in the lysosomal enzyme α-L-iduronidase (IDUA) due to a 3 bp deletion. An amphotropic retroviral vector (RV) expressing the canine IDUA was injected IV into newborn cats at 10(9) or 10(10) TU/kg. The canine protein differs from the feline protein at 130 positions of 653 aa. Expression can be followed by analysis of enzyme activity in serum, as some of the enzyme produced by cells is secreted into blood. Cats achieved high serum activity that averaged 286+/−416 (SD; N=7) U/ml and 2355+/−2763 (SD) U/ml at 1 month after gene transfer for the low and high RV dose, respectively. However, all animals lost expression by 3 months, which was associated with the development of cytotoxic T lymphocytes (CTLs) that could kill autologous cIDUA-expressing fibroblasts, and with a marked decrease in RV RNA sequences in the liver. In contrast, antibodies to cIDUA did not develop. In an attempt to block a CTL response, two cats were treated with the immunosuppressive agent CTLA4- Ig for 2 weeks around the time of transduction with the high dose of RV. CTLA4-Ig binds to CD80 or CD86, and prevents them from stimulating an immune response by activating CD28 on the surface of lymphocytes. These cats achieved 187+/−161 U/ml of IDUA activity in serum, which has been stable for 10 months to date, and did not develop a CTL response These data demonstrate that CTLA4-Ig can prevent a CTL response after neonatal gene therapy. Factors that reduce the efficiency of the immune system in newborn mice and/or humans include reduced secretion of cytokines by lymphocytes, and reduced levels of the co-stimulatory partners CD40 ligand and CD40. In contrast, lymphocytes from newborn humans have normal levels of CD80/CD86 and CD28. We hypothesize that the CD80/CD86 interaction with CD28 may be of particular importance in the newborn period in cats, explaining why transient CTLA4-Ig is so effective at inducing tolerance. The cat appears to be an excellent pre-clinical model for neonatal gene therapy.Molecular Therapy (2006) 13, S31–S31; doi: 10.1016/j.ymthe.2006.08.090 [ABSTRACT FROM AUTHOR]