Cytokine/Antibody Fusion Protein Design and Evaluation.

Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.

Gauging mixed climate extreme value distributions in tropical cyclone regions.

In tropical cyclone (TC) regions, tide gauge or numerical hindcast records are usually of insufficient length to have sampled sufficient cyclones to enable robust estimates of the climate of TC-induced extreme water level events. Synthetically-generated TC populations provide a means to define a broader set of plausible TC events to better define the probabilities associated with extreme water level events. The challenge is to unify the estimates of extremes from synthetically-generated TC populations with the observed records, which include mainly non-TC extremes resulting from tides and more frequently occurring atmospheric-depression weather and climate events. We find that extreme water level measurements in multiple tide gauge records in TC regions, some which span more than 100 years, exhibit a behaviour consistent with the combining of two populations, TC and non-TC. We develop an equation to model the combination of two populations of extremes in a single continuous mixed climate (MC) extreme value distribution (EVD). We then run statistical simulations to show that long term records including both historical and synthetic events can be better explained using MC than heavy-tailed generalised EVDs. This has implications for estimating extreme water levels when combining synthetic cyclone extreme sea levels with hindcast water levels to provide actionable information for coastal protection.

Projected incremental changes to extreme wind-driven wave heights for the twenty-first century.

Global climate change will alter wind sea and swell waves, modifying the severity, frequency and impact of episodic coastal flooding and morphological change. Global-scale estimates of increases to coastal impacts have been typically attributed to sea level rise and not specifically to changes to waves on their own. This study provides a reduced complexity method for applying projected extreme wave changes to local scale impact studies. We use non-stationary extreme value analysis to distil an incremental change signal in extreme wave heights and associate this with a change in the frequency of events globally. Extreme wave heights are not projected to increase everywhere. We find that the largest increases will typically be experienced at higher latitudes, and that there is high ensemble model agreement on an increase (doubling of events) for the waters south of Australia, the Arabian Sea and the Gulf of Guinea by the end of the twenty-first century.

Nomogram Predicting Bladder Cancer-specific Mortality After Neoadjuvant Chemotherapy and Radical Cystectomy for Muscle-invasive Bladder Cancer: Results of an International Consortium.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) is associated with improved overall and cancer-specific survival. The post-NAC pathological stage has previously been reported to be a major determinant of outcome. OBJECTIVE: To develop a postoperative nomogram for survival based on pathological and clinical parameters from an international consortium. DESIGN, SETTING, AND PARTICIPANTS: Between 2000 and 2015, 1866 patients with MIBC were treated at 19 institutions in the USA, Canada, and Europe. Analysis was limited to 640 patients with adequate follow-up who had received three or more cycles of NAC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A nomogram for bladder cancer-specific mortality (BCSM) was developed by multivariable Cox regression analysis. Decision curve analysis was used to assess the model's clinical utility. RESULTS AND LIMITATIONS: A total of 640 patients were identified. Downstaging to non-MIBC (ypT1, ypTa, and ypTis) occurred in 271 patients (42 %), and 113 (17 %) achieved a complete response (ypT0N0). The 5-yr BCSM was 47.2 % (95 % confidence interval [CI]: 41.2-52.6 %). On multivariable analysis, covariates with a statistically significant association with BCSM were lymph node metastasis (hazard ratio [HR] 1.90 [95% CI: 1.4-2.6]; p < 0.001), positive surgical margins (HR 2.01 [95 % CI: 1.3-2.9]; p < 0.001), and pathological stage (with ypT0/Tis/Ta/T1 as reference: ypT2 [HR 2.77 {95 % CI: 1.7-4.6}; p < 0.001] and ypT3-4 [HR 5.9 {95 % CI: 3.8-9.3}; p < 0.001]). The area under the curve of the model predicting 5-yr BCSM after cross validation with 300 bootstraps was 75.4 % (95 % CI: 68.1-82.6 %). Decision curve analyses showed a modest net benefit for the use of the BCSM nomogram in the current cohort compared with the use of American Joint Committee on Cancer staging alone. Limitations include the retrospective study design and the lack of central pathology. CONCLUSIONS: We have developed and internally validated a nomogram predicting BCSM after NAC and radical cystectomy for MIBC. The nomogram will be useful for patient counseling and in the identification of patients at high risk for BCSM suitable for enrollment in clinical trials of adjuvant therapy. PATIENT SUMMARY: In this report, we looked at the outcomes of patients with muscle-invasive bladder cancer in a large multi-institutional population. We found that we can accurately predict death after radical surgical treatment in patients treated with chemotherapy before surgery. We conclude that the pathological report provides key factors for determining survival probability.

Concomitant CIS on TURBT does not impact oncological outcomes in patients treated with neoadjuvant or induction chemotherapy followed by radical cystectomy.

BACKGROUND: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle invasive bladder cancer improves all-cause and cancer specific survival. We aimed to evaluate whether the detection of carcinoma in situ (CIS) at the time of initial transurethral resection of bladder tumor (TURBT) has an oncological impact on the response to NAC prior to radical cystectomy. PATIENTS AND METHODS: Patients were identified retrospectively from 19 centers who received at least three cycles of NAC or induction chemotherapy for cT2-T4aN0-3M0 urothelial carcinoma of the bladder followed by radical cystectomy between 2000 and 2013. The primary and secondary outcomes were pathological response and overall survival, respectively. Multivariable analysis was performed to determine the independent predictive value of CIS on these outcomes. RESULTS: Of 1213 patients included in the analysis, 21.8% had concomitant CIS. Baseline clinical and pathologic characteristics of the 'CIS' versus 'no-CIS' groups were similar. The pathological response did not differ between the two arms when response was defined as pT0N0 (17.9% with CIS vs 21.9% without CIS; p = 0.16) which may indicate that patients with CIS may be less sensitive to NAC or ≤ pT1N0 (42.8% with CIS vs 37.8% without CIS; p = 0.15). On Cox regression model for overall survival for the cN0 cohort, the presence of CIS was not associated with survival (HR 0.86 (95% CI 0.63-1.18; p = 0.35). The presence of LVI (HR 1.41, 95% CI 1.01-1.96; p = 0.04), hydronephrosis (HR 1.63, 95% CI 1.23-2.16; p = 0.001) and use of chemotherapy other than ddMVAC (HR 0.57, 95% CI 0.34-0.94; p = 0.03) were associated with shorter overall survival. For the whole cohort, the presence of CIS was also not associated with survival (HR 1.05 (95% CI 0.82-1.35; p = 0.70). CONCLUSION: In this multicenter, real-world cohort, CIS status at TURBT did not affect pathologic response to neoadjuvant or induction chemotherapy. This study is limited by its retrospective nature as well as variability in chemotherapy regimens and surveillance regimens.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

The benefits of newborn screening for cystic fibrosis: The Canadian experience.

BACKGROUND: The impact of newborn screening (NBS) for cystic fibrosis (CF) on early indicators of long-term health was evaluated in the context of government-sponsored healthcare and access to current therapies. METHODS: Using data from the Canadian CF Registry between 2008 and 2013, we compared the rates of respiratory infections and markers of nutritional status in those diagnosed through NBS to those who were diagnosed clinically within the same time period using Mann-Whitney and Fischer's exact test as appropriate. RESULTS: The study included 303 subjects, 201 in the NBS group and 102 in the non-NBS group. NBS patients were diagnosed earlier and had their first clinic visit at a younger age. Pancreatic insufficiency was less common in NBS patients. The incidence of Pseudomonas aeruginosa and Staphylococcus aureus were lower in NBS patients. After adjusting for age at clinic visit, gender, pancreatic status, and Pseudomonas aeruginosa infection status, mean z-scores for weight-for-age and height-for-age were higher in NBS patients, with no differences in BMI-for-age. CONCLUSIONS: NBS programs for CF lead to improved long-term health outcomes for the CF population.

Transgenic Virus Resistance in Crop-Wild Cucurbita pepo Does Not Prevent Vertical Transmission of Zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) is an economically important pathogen of cucurbits that is transmitted both horizontally and vertically. Although ZYMV is seed-transmitted in Cucurbita pepo, the potential for seed transmission in virus-resistant transgenic cultivars is not known. We crossed and backcrossed a transgenic squash cultivar with wild C. pepo, and determined whether seed-to-seedling transmission of ZYMV was possible in seeds harvested from transgenic backcrossed C. pepo. We then compared these transmission rates to those of non-transgenic (backcrossed and wild) C. pepo. The overall seed-to-seedling transmission rate in ZYMV was similar to those found in previous studies (1.37%), with no significant difference between transgenic backcrossed (2.48%) and non-transgenic (1.03%) backcrossed and wild squash. Fewer transgenic backcrossed plants had symptom development (7%) in comparison with all non-transgenic plants (26%) and may be instrumental in preventing yield reduction due to ZYMV. Our study shows that ZYMV is seed transmitted in transgenic backcrossed squash, which may affect the spread of ZYMV via the movement of ZYMV-infected seeds. Deep genome sequencing of the seed-transmitted viral populations revealed that 23% of the variants found in this study were present in other vertically transmitted ZYMV populations, suggesting that these variants may be necessary for seed transmission or are distributed geographically via seeds.

Cystic fibrosis (cf) and ovarian reserve: A cross-sectional study examining serum anti-mullerian hormone (amh) in young women.

BACKGROUND: Reduced female fertility in CF is believed to be due to thick cervical mucous, poor nutritional status, functional hypogonadotropic hypogonadism and possibly increased inflammation. Literature suggests that reduced ovarian reserve may also play a role. METHODS: 20 women with CF and 20 controls age 18-35 years were recruited. Serum anti-mullerian hormone (AMH), estradiol and follicle stimulating hormone (FSH) were assessed as well as antral follicle count (AFC) using transvaginal ultrasound. RESULTS: Women with CF had significantly lower AMH levels than controls (17.8+/-4.7 vs. 33.2+/-21.0 pmol/L respectively; p=0.004). There were no differences in estradiol, FSH or AFC. CONCLUSIONS: Women with CF have reduced ovarian reserve which may contribute to sub-fertility. CF care providers should consider referring women with CF to fertility specialists early to optimize chances of pregnancy.

Personalizing, not patronizing: the case for patient autonomy by unbiased presentation of management options in stage I testicular cancer.

Testicular cancer (TC) is the most common neoplasm in males aged 15-40 years. The majority of patients have no evidence of metastases at diagnosis and thus have clinical stage I (CSI) disease [Oldenburg J, Fossa SD, Nuver J et al. Testicular seminoma and non-seminoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2013; 24(Suppl 6): vi125-vi132; de Wit R, Fizazi K. Controversies in the management of clinical stage I testis cancer. J Clin Oncol 2006; 24: 5482-5492.]. Management of CSI TC is controversial and options include surveillance and active treatment. Different forms of adjuvant therapy exist, including either one or two cycles of carboplatin chemotherapy or radiotherapy for seminoma and either one or two cycles of cisplatin-based chemotherapy or retroperitoneal lymph node dissection for non-seminoma. Long-term disease-specific survival is ∼99% with any of these approaches, including surveillance. While surveillance allows most patients to avoid additional treatment, adjuvant therapy markedly lowers the relapse rate. Weighing the net benefits of surveillance against those of adjuvant treatment depends on prioritizing competing aims such as avoiding unnecessary treatment, avoiding more burdensome treatment with salvage chemotherapy and minimizing the anxiety, stress and life disruption associated with relapse. Unbiased information about the advantages and disadvantages of surveillance and adjuvant treatment is a prerequisite for informed consent by the patient. In a clinical scenario like CSI TC, where different disease-management options produce indistinguishable long-term survival rates, patient values, priorities and preferences should be taken into account. In this review, we provide an overview about risk factors for relapse, potential benefits and harms of adjuvant chemotherapy and active surveillance and a rationale for involving patients in individualized decision making about their treatment rather than adopting a uniform recommendation for all.

Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine.

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.

Dynamics of short- and long-term association between a bacterial plant pathogen and its arthropod vector.

The dynamics of association between pathogens and vectors can strongly influence epidemiology. It has been proposed that wilt disease epidemics in cucurbit populations are sustained by persistent colonization of beetle vectors (Acalymma vittatum) by the bacterial phytopathogen Erwinia tracheiphila. We developed a qPCR method to quantify E. tracheiphila in whole beetles and frass and used it to assess pathogen acquisition and retention following variable exposure to infected plants. We found that (i) E. tracheiphila is present in frass in as little as three hours after feeding on infected plants and can be transmitted with no incubation period by vectors given brief exposure to infected plants, but also by persistently colonized vectors several weeks following exposure; (ii) duration of exposure influences rates of long-term colonization; (iii) frass infectivity (assessed via inoculation experiments) reflects bacterial levels in frass samples across time; and (iv) vectors rarely clear E. tracheiphila infections, but suffer no apparent loss of fitness. These results describe a pattern conducive to the effective maintenance of E. tracheiphila within cucurbit populations.

Zucchini yellow mosaic virus (ZYMV, Potyvirus): vertical transmission, seed infection and cryptic infections.

The role played by seed transmission in the evolution and epidemiology of viral crop pathogens remains unclear. We determined the seed infection and vertical transmission rates of zucchini yellow mosaic virus (ZYMV), in addition to undertaking Illumina sequencing of nine vertically transmitted ZYMV populations. We previously determined the seed-to-seedling transmission rate of ZYMV in Cucurbita pepo ssp. texana (a wild gourd) to be 1.6%, and herein observed a similar rate (1.8%) in the subsequent generation. We also observed that the seed infection rate is substantially higher (21.9%) than the seed-to-seedling transmission rate, suggesting that a major population bottleneck occurs during seed germination and seedling growth. In contrast, that two thirds of the variants present in the horizontally transmitted inoculant population were also present in the vertically transmitted populations implies that the bottleneck at vertical transmission may not be particularly severe. Strikingly, all of the vertically infected plants were symptomless in contrast to those infected horizontally, suggesting that vertical infection may be cryptic. Although no known virulence determining mutations were observed in the vertically infected samples, the 5' untranslated region was highly variable, with at least 26 different major haplotypes in this region compared to the two major haplotypes observed in the horizontally transmitted population. That the regions necessary for vector transmission are retained in the vertically infected populations, combined with the cryptic nature of vertical infection, suggests that seed transmission may be a significant contributor to the spread of ZYMV.

Prognostic relevance of dynamic hyperinflation during cardiopulmonary exercise testing in adult patients with cystic fibrosis.

BACKGROUND: Dynamic hyperinflation during cardiopulmonary exercise testing (CPET) in cystic fibrosis (CF) has not been well characterized, and little is known regarding its prevalence, risk factors and clinical associations. METHODS: CPET data from 109 adult patients with mild-to-moderate CF was used, in this retrospective study, to characterize and determine the prevalence of dynamic hyperinflation, and evaluate its relationship with lung function and exercise tolerance, clinical symptoms, and prognosis over a two-year period. RESULTS: 58% of patients responded to CPET with dynamic hyperinflation. These patients had significantly lower lung function (FEV1 66±19 versus 79±18%pred., p<0.01) and exercise tolerance (peak oxygen uptake 28.7±8.1 versus 32.9±6.1 mL·kg(-1)·min(-1), p=0.02), and experienced greater shortness of breath at peak exercise (7±3 versus 5±2 Modified Borg scale, p=0.04) compared to patients who responded without dynamic hyperinflation. Significant relationships between FEV1, FVC, FEV1/FVC, FEF(25-75) and dynamic hyperinflation were shown (p<0.01; p=0.02; p<0.01; p<0.01, respectively). Dynamic hyperinflation was also significantly correlated with oxygen uptake, tidal volume, work-rate and shortness of breath at peak exercise (p=0.03; p<0.01; p<0.01; p=0.04, respectively). Responding to CPET with or without dynamic hyperinflation did not significantly predict FEV1 at 2 years beyond the FEV1 at baseline (p=0.06), or increase the likelihood of experiencing a pulmonary exacerbation over a two-year period (p=0.24). CONCLUSION: The prevalence of dynamic hyperinflation during CPET in adult patients with mild-to-moderate CF is high, and is associated with reduced lung function and exercise tolerance, and increased exertional dyspnea. However, identifying dynamic hyperinflation during CPET had limited prognostic value for lung function and pulmonary exacerbation.

Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O(2) tension-induced ATP release.

Statin drugs inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which reduces the synthesis of both cholesterol and isoprenoids (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), with the latter being lipid molecules responsible for the posttranslational modification of small GTP-binding proteins such as Rho. Effects of statins, independent of lowering blood cholesterol levels, are thought to occur by inhibition of Rho/Rho kinase. The Rho kinase inhibitor Y-27632 has been reported to increase both erythrocyte deformability and low O2 tension-induced ATP release. Here, we tested the hypothesis that by inhibiting Rho/Rho kinase, simvastatin would increase both erythrocyte deformability and low O2 tension-induced ATP release. Male Sprague-Dawley rats were divided into two groups, control or simvastatin treated [simvastatin-supplemented chow (0.02%)], for 4 wk. Simvastatin treatment increased rat erythrocyte deformability compared with controls (n = 6, P < 0.05). However, erythrocytes of simvastatin-treated rats (n = 9, P < 0.05) exhibited impaired low O2 tension-induced ATP release. Similarly, the geranylgeranyl transferase inhibitor GGTI-2133 (10 µM) also increased deformability and impaired low O2 tension-induced ATP release in healthy human erythrocytes (P < 0.05). Interestingly, ATP release in response to mastoparan 7 (n = 7, P < 0.05), which directly activates Gi, and isoproterenol (n = 5, P < 0.05), which signals through Gs, was not altered by incubation with GGTI-2133. These results suggest that although statins increase erythrocyte deformability, likely by inhibiting geranylgeranylation, the finding that both statins and a geranylgeranyl transferase inhibitor attenuated low O2 tension-induced ATP release demonstrates that factors in addition to erythrocyte deformability are critical for ATP release in response to this physiological stimulus.

Magnetic resonance imaging for the study of ovarian follicles in the mouse.

Additional tools to analyze follicle development would be highly advantageous because current methods require sacrifice of animals at specific times and time-consuming sectioning of tissues for histologic analysis. Magnetic resonance imaging (MRI) may provide a less involved, faster and more cost-effective method to analyze follicles in whole ovaries. Fixed ovaries were collected at different stages of the estrus cycle and after stimulation with gonadotrophins (24 and 48 h post pregnant mares serum (PMSG), and 10 and 24 h post human chorionic gonadotrophin (hCG)) with or without administration of the contrast agent gadodiamide. The MR images were generated using a vertical-bore, 11.7 Tesla MR system. Analysis of the MR images revealed large antral follicles in fixed ovaries with the oocyte and cumulus mass identifiable within preovulatory follicles. The use of gadodiamide had no impact on the quality of MR images obtained. The fixed ovaries were paraffin embedded, sectioned, and hematoxylin stained. Follicles were counted using the MR images and the histology sections. Preovulatory follicle numbers determined using MR images were comparable to those using histology; however counts of smaller follicles were inconsistent. MRI of gonadotrophin-stimulated ovaries in situ did not reveal discernable ovarian structures. Therefore, MRI is a useful tool for studying whole fixed ovaries leaving the ovary intact for additional analyses or for selection of samples based on morphology. The MRI is also useful for identifying preovulatory follicles, although analysis of smaller follicles is not possible, and thus the potential exists for cyst analysis in mouse models of polycystic ovarian syndrome (PCOS).