IL-27-engineered CAR.19-NK-92 cells exhibit enhanced therapeutic efficacy.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies.

SARS-CoV2 entry factors are expressed in primary human glioblastoma and recapitulated in cerebral organoid models.

PURPOSE: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults with a median overall survival of only 14.6 months despite aggressive treatment. While immunotherapy has been successful in other cancers, its benefit has been proven elusive in GBM, mainly due to a markedly immunosuppressive tumor microenvironment. SARS-CoV-2 has been associated with the development of a pronounced central nervous system (CNS) inflammatory response when infecting different cells including astrocytes, endothelial cells, and microglia. While SARS-CoV2 entry factors have been described in different tissues, their presence and implication on GBM aggressiveness or microenvironment has not been studied on appropriate preclinical models. METHODS: We evaluated the presence of crucial SARS-CoV-2 entry factors: ACE2, TMPRSS2, and NRP1 in matched surgically-derived GBM tissue, cells lines, and organoids; as well as in human brain derived specimens using immunohistochemistry, confocal pixel line intensity quantification, and transcriptome analysis. RESULTS: We show that patient derived-GBM tissue and cell cultures express SARS-CoV2 entry factors, being NRP1 the most crucial facilitator of SARS-CoV-2 infection in GBM. Moreover, we demonstrate that, receptor expression remains present in our GBM organoids, making them an adequate model to study the effect of this virus in GBM for the potential development of viral therapies in the future. CONCLUSION: Our findings suggest that the SARS-CoV-2 virus entry factors are expressed in primary tissues and organoid models and could be potentially utilized to study the susceptibility of GBM to this virus to target or modulate the tumor microenviroment.

Immune checkpoint inhibition and single fraction stereotactic radiosurgery in brain metastases from non-small cell lung cancer: an international multicenter study of 395 patients.

PURPOSE: Approximately 80% of brain metastases originate from non-small cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICI) and stereotactic radiosurgery (SRS) are frequently utilized in this setting. However, concerns remain regarding the risk of radiation necrosis (RN) when SRS and ICI are administered concurrently. METHODS: A retrospective study was conducted through the International Radiosurgery Research Foundation. Logistic regression models and competing risks analyses were utilized to identify predictors of any grade RN and symptomatic RN (SRN). RESULTS: The study included 395 patients with 2,540 brain metastases treated with single fraction SRS and ICI across 11 institutions in four countries with a median follow-up of 14.2 months. The median age was 67 years. The median margin SRS dose was 19 Gy; 36.5% of patients had a V12 Gy ≥ 10 cm3. On multivariable analysis, V12 Gy ≥ 10 cm3 was a significant predictor of developing any grade RN (OR: 2.18) and SRN (OR: 3.95). At 1-year, the cumulative incidence of any grade and SRN for all patients was 4.8% and 3.8%, respectively. For concurrent and non-concurrent groups, the cumulative incidence of any grade RN was 3.8% versus 5.3%, respectively (p = 0.35); and for SRN was 3.8% vs. 3.6%, respectively (p = 0.95). CONCLUSION: The risk of any grade RN and symptomatic RN following single fraction SRS and ICI for NSCLC brain metastases increases as V12 Gy exceeds 10 cm3. Concurrent ICI and SRS do not appear to increase this risk. Radiosurgical planning techniques should aim to minimize V12 Gy.

Wound inflammation post-orchiectomy affects the social dynamic of Nelore bulls.

BACKGROUND: Confinement of cattle imposes spatial restrictions and predisposes to aversive social encounters that can lead to contusions, wounds, pain, stress, fright, and reduced productivity. Although endogenous testosterone concentrations are linked to agonistic dominance behaviors in males, it is unknown whether decreased blood testosterone concentrations after castration alter social hierarchy rank in Nelore bulls. Therefore, in this study, we investigated the impact of the surgical would inflammation post-orchiectomy on social dynamics in a group of Nelore bulls (Bos indicus). Fourteen Nelore (Bos indicus) bulls were castrated and assessed pre- and post-surgically. Parameters evaluated were agonistic (mounting, headbutting, and fighting) and affiliative (head-play) behavior, plasma testosterone concentrations, average daily weight gain (ADG), and a score for severity of post-surgical infection. Exploratory statistics included social network analysis (SNA), hierarchy rank delta (Δ), and principal component analysis (PCA). Furthermore, statistical inferences included the Wilcoxon test, multiple logistic regression models, and Spearman's correlation. RESULTS: The social dynamic of Nelore bulls was modified after castration based on the findings of the SNA and the PCA. The moderate correlation between the postoperative inflammation level with the Δ, and the significant effect of this level in the logistic model post-castration were partially attributed to effects of pain on social relations. CONCLUSIONS: Our findings suggest the severity of post-surgical inflammation, which has an association with pain intensity, was closely associated with changes in the social hierarchy.

Absence of the torcular, review of venous sinus anatomy, and the simplified dural sinus classification.

BACKGROUND: Classically, the torcular Herophili is described as the symmetric junction between the superior sagittal sinus (SSS), transverse sinuses (TSs), and straight sinus (SS). However, finding this pattern in practice is not standard. Anatomical variations are common, and different drainage patterns should be expected. Existing literature proposes highly detailed descriptions and classifications of this region. Still, a simplified and practical categorization is not available. METHODS: We present an anatomical finding of the torcular Herophili discovered on a cadaveric dissection. Then, we conducted a retrospective study examining the 100 most recent cranial magnetic resonance venographies (MRVs) from the Mayo Clinic, labeling them with a new proposed dural sinus classification system. Images were initially classified by two authors and further validated by a board-certified neurosurgeon and a board-certified neuroradiologist from our institution. To measure consistency in image identification, two additional international neurosurgeons were asked to classify a subset of the same MRV images, and their answers were compared. RESULTS: Of the MRV cohort, 33 patients were male and 67 were female. Their ages ranged from 18 to 86 years, with a mean of 47.35 years and a median of 49 years. Upon examination, 53 patients presented as confluent (53%), 9 as SSS divergent (9%), 25 as SS divergent (25%), 11 as circular (11%), and 2 as trifurcated (2%). The inter-rater reliability ranked very good; agreement between the two neurosurgeons was 83% (κ = 0.830, p < 0.0005). CONCLUSION: The confluence of the venous sinuses is a highly variable anatomical area that is rarely evaluated with neuroimaging before surgery. The classic textbook configuration is not the rule. Using a simplified classification system may increase awareness and hopefully patient safety by preparing the physician for anatomical variations that they will encounter in a surgical or clinical scenario.

Strengthening public health policies for childhood cancer: Peru's achievements through the WHO Global Initiative for Childhood Cancer.

Objective: To report the progress in Peru, since June 2019, in the implementation of the World Health Organization Global Initiative for Childhood Cancer using the CureAll framework, which can be replicated in low- and middle-income countries. Methods: A mixed method was used of participatory and documentary evaluation. The participatory evaluation included stakeholders from various government institutions, nonprofit organizations, and international partners. The documentary aspect consisted of a review of data on the regulatory environment, national projects, and interventions implemented. The Ministry of Health engaged more than 150 participants to form working committees, which have developed policy and regulatory documents to strengthen care services. Results: Achievements include a decrease in the national treatment abandonment rate from 18.6% to 8.5%, the approval of the Childhood Cancer Law, improvements in the management of patients with febrile neutropenia, and a reduction in rates of events of clinical deterioration and mortality of hospitalized patients. The Cure All implementation framework allows local teams to implement specific strategies and monitor early outcomes in pediatric oncology. Conclusions: The results obtained reflect the teamwork, the leadership of the authorities, the technical support of professionals, and the support of involved organizations. Further actions will be needed to guarantee sustainability, and monitoring tools are needed to assure success in the planned activities.

Radiation necrosis in renal cell carcinoma brain metastases treated with checkpoint inhibitors and radiosurgery: An international multicenter study.

BACKGROUND: Patients with renal cell carcinoma (RCC) brain metastases are frequently treated with immune checkpoint inhibitors (ICIs) and stereotactic radiosurgery (SRS). However, data reporting on the risk of developing radiation necrosis (RN) are limited. METHODS: RN rates were compared for concurrent therapy (ICI/SRS administration within 4 weeks of one another) and nonconcurrent therapy with the χ2 test. Univariable logistic regression was used to identify factors associated with developing RN. RESULTS: Fifty patients (23 concurrent and 27 nonconcurrent) with 395 brain metastases were analyzed. The median follow-up was 12.1 months; the median age was 65 years. The median margin dose was 20 Gy, and 4% underwent prior whole-brain radiation therapy (WBRT). The median treated tumor volume was 3.32 cm3 (range, 0.06-42.38 cm3 ); the median volume of normal brain tissue receiving a dose of 12 Gy or higher (V12 Gy) was 8.42 cm3 (range, 0.27-111.22 cm3 ). Any-grade RN occurred in 17.4% and 22.2% in the concurrent and nonconcurrent groups, respectively (P = .67). Symptomatic RN occurred in 4.3% and 14.8% in the concurrent and nonconcurrent groups, respectively (P = .23). Increased tumor volume during SRS (odds ratio [OR], 1.08; 95% confidence interval [CI], 1.01-1.19; P = .04) was associated with developing RN, although V12 Gy (OR, 1.03; 95% CI, 0.99-1.06; P = .06), concurrent therapy (OR, 0.74; 95% CI, 0.17-2.30; P = .76), prior WBRT, and ICI agents were not statistically significant. CONCLUSIONS: Symptomatic RN occurs in a minority of patients with RCC brain metastases treated with ICI/SRS. The majority of events were grade 1 to 3 and were managed medically. Concurrent ICI/SRS does not appear to increase this risk. Attempts to improve dose conformality (reduce V12) may be the most successful mitigation strategy in single-fraction SRS.

The prediction of Metabolic Syndrome alterations is improved by combining waist circumference and handgrip strength measurements compared to either alone.

BACKGROUND: Adiposity is a major component of the metabolic syndrome (MetS), low muscle strength has also been identified as a risk factor for MetS and for cardiovascular disease. We describe the prevalence of MetS and evaluate the relationship between muscle strength, anthropometric measures of adiposity, and associations with the cluster of the components of MetS, in a middle-income country. METHODS: MetS was defined by the International Diabetes Federation criteria. To assess the association between anthropometric variables (waist circumference (WC), waist-to-hip ratio (W/H), body mass index (BMI)), strength (handgrip/kg bodyweight (HGS/BW)) and the cluster of MetS, we created a MetS score. For each alteration (high triglycerides, low HDLc, dysglycemia, or high blood pressure) one point was conferred. To evaluate the association an index of fat:muscle and MetS score, participants were divided into 9 groups based on combinations of sex-specific tertiles of WC and HGS/BW. RESULTS: The overall prevalence of MetS in the 5,026 participants (64% women; mean age 51.2 years) was 42%. Lower HGS/BW, and higher WC, BMI, and W/H were associated with a higher MetS score. Amongst the 9 HGS/BW:WC groups, participants in the lowest tertile of HGS/BW and the highest tertile of WC had a higher MetS score (OR = 4.69 in women and OR = 8.25 in men;p < 0.01) compared to those in the highest tertile of HGS/BW and in the lowest tertile of WC. CONCLUSION: WC was the principal risk factor for a high MetS score and an inverse association between HGS/BW and MetS score was found. Combining these anthropometric measures improved the prediction of metabolic alterations over either alone.

The evolution of stereotactic radiosurgery in neurosurgical practice.

INTRODUCTION: Stereotactic radiosurgery (SRS) was born in an attempt to treat complex intracranial pathologies in a fashion whereby open surgery would create unnecessary or excessive risk. To create this innovation, it was necessary to harness advances in other fields such as engineering, physics, radiology, and computer science. METHODS: We review the history of SRS to provide context to today's current state, as well as guide future advancement in the field. RESULTS: Since time of Lars Leksell, the young Swedish neurosurgeon who pioneered the development of the SRS, the collegial and essential partnership between neurosurgeons, radiation oncologists and physicists has given rise to radiosurgery as a prominent and successful tool in neurosurgical practice. CONCLUSION: We examine how neurosurgeons have helped foster the SRS evolution and how this evolution has impacted neurosurgical practice as well as that of radiation oncology and neuro-oncology.

Adipose-Derived Stem Cells Therapy for Radiation-Induced Skin Injury.

BACKGROUND: Radiation-induced skin injuries have been treated with different medical therapies and have shown diverse outcomes. We aim to evaluate the effect of adipose-derived stem cells (ADSCs) therapy on radiation-induced skin injury. METHODS: We performed a review by querying PubMed, Ovid MEDLINE, and EMBASE databases from inception to April 2020 following Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. The MeSH terms "adipose-derived stem cells," "wound healing," "radiation," and synonyms in combinations determined our search strategy. Experimental peer-reviewed articles describing the protocol and comparing the results with controls were included. Non-English studies were excluded. RESULTS: Our search recorded a total of 137 articles. Only 8 studies met our inclusion criteria and were included in this review. Five studies evaluated the use of ADSC alone, whereas the others evaluated the efficacy of ADSC seeded in scaffolds. Adipose-derived stem cell-based therapies, either alone or seeded in scaffolds, were shown to improve wound healing in most studies when compared with controls. CONCLUSIONS: There is evidence supporting the positive benefits from ADSC-based therapies in radiation-induced skin injury. However, further studies are needed to standardize the method of ADSC extraction, radiation-induced skin injury experimental model, and increase the time of follow-up to evaluate the results accurately.

Modified Cranioplasty and Latissimus Dorsi Flap for an Exophytic Invasive Brain Tumor.

ABSTRACT: We describe the clinical case of a 62-year-old man with an atypical grade II meningioma, invading bone and scalp, and present a step-by-step video description of a modified technique for cranioplasty and scalp reconstruction with latissimus dorsi flap. By using this technique, we aim to minimize the space between the dura and the flap, which would decrease the risk of fluid collections and infections.

Novel Strategies for Nanoparticle-Based Radiosensitization in Glioblastoma.

Radiotherapy (RT) is one of the cornerstones in the current treatment paradigm for glioblastoma (GBM). However, little has changed in the management of GBM since the establishment of the current protocol in 2005, and the prognosis remains grim. Radioresistance is one of the hallmarks for treatment failure, and different therapeutic strategies are aimed at overcoming it. Among these strategies, nanomedicine has advantages over conventional tumor therapeutics, including improvements in drug delivery and enhanced antitumor properties. Radiosensitizing strategies using nanoparticles (NP) are actively under study and hold promise to improve the treatment response. We aim to describe the basis of nanomedicine for GBM treatment, current evidence in radiosensitization efforts using nanoparticles, and novel strategies, such as preoperative radiation, that could be synergized with nanoradiosensitizers.

Intraventricular choroid plexus tumors: clinical characteristics and impact of current management on survival.

INTRODUCTION: Choroid plexus tumors (CPTs) represent one of the most common intraventricular tumors. Although most are benign, they often reach considerable sizes before clinical manifestation, challenging their surgical management. We aim to describe the clinical characteristics and the impact of current management on the survival of patients harboring intraventricular CPT. METHODS: The National Cancer Database (NCDB) was queried to identify biopsy-proven intraventricular CPT patients (2004-2015). Demographic and patterns of care were described, the log-rank method was used to independently analyze survival according to age, WHO grade and extent of resection (EOR). Multivariate analysis was performed to investigate the impact of prognostic factors on overall survival (OS). RESULTS: A total of 439 CPT patients with known WHO grade were included. WHO grade I tumors were more frequent in adults, while WHO grade III tumors were more common in pediatric population. Most CPTs were benign, with a median tumor size of 3-4 cm. Mean tumor size in pediatric population was greater than in adult population (4.39 cm vs. 2.7 cm; p < 0.01). Frequency was similar between males and females (51.7% vs. 48.3%; p > 0.0.5). Five- and ten-year OS among all patients was 87% and 84%, respectively. EOR was not associated with survival for any WHO grade. On multivariable analysis, only patient age (p = 0.022), WHO grade (p = 0.003) and medical comorbidity scores (p = 0.002) were independently associated with OS after diagnosis. CONCLUSION: Patients with CPTs present at different stages of life, with sizable tumor burden and distinct WHO grade prevalence. Considering their favorable survival, efforts to improve tumor control should be meticulously weighed against the long-term risk associated with surgery, radiation, and chemotherapy.

May Measurement Month 2018: an analysis of blood pressure screening results from Colombia.

High blood pressure (BP) is the leading global preventable cause of death and the most common risk factor for cardiovascular disease (CVD). However, due to its asymptomatic nature, the lack of awareness of this condition causes underdiagnosis and low rates of adherence to pharmacological treatment. Looking for practical approaches to increase awareness worldwide, the International Society of Hypertension (ISH) implemented the 2nd May Measurement Month campaign in 2018 (MMM18). In order to contribute to this initiative, Colombia participated as one of the 89 countries involved in this hypertension screening programme. Blood pressure was measured in subjects from 11 departments in Colombia. Under the leadership of the Fundación Oftalmológica de Santander (FOSCAL), 400 volunteers across the country collected the data following the MMM protocol. Measurements from 35 548 participants with a mean age of 41.9 years were obtained. In total, 9475 (26.7%) of the total population studied had hypertension. Of those with hypertension, 69.9% of these subjects were aware of their condition, 65.0% were on antihypertensive medication, and 43.1% had controlled BP. Of those on medication, 66.3% had controlled BP. Hypertension screening, awareness, treatment, and control should be a priority in public health objectives due to its elevated burden of disease and direct association with increased CVD. The MMM campaign provided a positive impact in the diagnosis of hypertension across Colombia. Although efforts are being made to expand treatment capability and adherence, still more are needed to insure a broader coverage of antihypertensive medication in Colombia.

Awake vs. asleep motor mapping for glioma resection: a systematic review and meta-analysis.

BACKGROUND: Intraoperative stimulation (IS) mapping has become the preferred standard treatment for eloquent tumors as it permits a more accurate identification of functional areas, allowing surgeons to achieve higher extents of resection (EOR) and decrease postoperative morbidity. For lesions adjacent to the perirolandic area and descending motor tracts, mapping can be done with both awake craniotomy (AC) and under general anesthesia (GA). OBJECTIVE: We aimed to determine which anesthetic protocol-AC vs. GA-provides better patient outcomes by comparing EOR and postoperative morbidity for surgeries using IS mapping in gliomas located near or in motor areas of the brain. METHODS: A systematic literature search was carried out to identify relevant studies from 1983 to 2019. Seven databases were screened. A total of 2351 glioma patients from 17 studies were analyzed. RESULTS: A random-effects meta-analysis revealed a trend towards a higher mean EOR in AC [90.1% (95% C.I. 85.8-93.8)] than with GA [81.7% (95% C.I. 72.4-89.7)] (p = 0.06). Neurological deficits were divided by timing and severity for analysis. There was no significant difference in early neurological deficits [20.9% (95% C.I. 4.1-45.0) vs. 25.4% (95% C.I. 13.6-39.2)] (p = 0.74), late neurological deficits [17.1% (95% C.I. 0.0-50.0) vs. 3.8% (95% C.I. 1.1-7.6)] (p = 0.06), or in non-severe [28.4% (95% C.I. 0.0-88.5) vs. 20.1% (95% C.I. 7.1-32.2)] (p = 0.72), and severe morbidity [2.6% (95% C.I. 0.0-15.5) vs. 4.5% (95% C.I. 1.1-9.6)] (p = 0.89) between patients who underwent AC versus GA, respectively. CONCLUSION: Mapping during resection of gliomas located in or near the perirolandic area and descending motor tracts can be safely carried out with both AC and GA.

May Measurement Month 2017: an analysis of blood pressure screening results in Colombia-Americas.

Despite the availability of efficient methods to evaluate blood pressure (BP) and of safe and efficient medication to treat and control hypertension, the levels of awareness, treatment and control are very low globally, particularly in low- and middle-income countries. To highlight the importance of improving these rates, the International Society of Hypertension (ISH) endorsed by the World Hypertension League have implemented the May Measurement Month initiative. We present here the results obtained in Colombia. The Fundación Oftalmológica de Santander (FOSCAL) led the implementation of this strategy in Colombia and 11 departments participated. The data collection followed the guidelines of the ISH. The information collected was compiled for the report generation and the submission to the Technical Secretariat of the ISH. Data cleaning was performed locally by FOSCAL. Data were collated and analysed centrally. A total of 22 258 participants (58.8% female) were included in the analysis. Mean age was 40.9 ± 17.7 years. Age and sex-standardized BP excluding participants receiving BP medications was 118/74.3 mmHg, and in those on treatment 125/78 mmHg. High BP was present in 5036 (22.8%) individuals, 1637 of 18 644 (8.8%) who were not receiving anti-hypertensive medications were hypertensive, and 961 of 3359 (28.6%) receiving treatment were not controlled. These results highlight the need to develop innovative promotion strategies at individual and population levels to increase the awareness of the importance of BP, and the consequences of not having well-controlled hypertension. This initiative is an effective and easy to implement strategy that should be maintained in the coming years.

Stereotactic radiosurgery for brain metastases from malignant melanoma and the impact of hemorrhagic metastases.

INTRODUCTION: Stereotactic radiosurgery (SRS) is a common treatment modality among patients with brain metastases, particularly from malignant melanoma. Our objective was to investigate the difference in local control, toxicity, and survival among patients with hemorrhagic and solid melanoma brain metastases. METHODS: We collected demographic, treatment, local control, toxicity, and survival for 134 patients with a total of 936 intracranial melanoma metastases who underwent SRS between 1998 and 2015. Pre-radiosurgical diagnostic imaging was reviewed for evidence of hemorrhage (melanin-containing or clearly hemorrhagic). RESULTS: The cohort consisted of 92 men and 42 women with a mea age of 61.7 years (range 21.2-84.9) at the time of radiosurgery. Overall survival of patients with brain metastases from malignant melanoma was 42, 31, 12% at 12, 24, and 72 months from date of first SRS. At 6 months, 43% of the patients with hemorrhagic metastases had local tumor control compared to 83% of solid melanoma metastases (p < 0.001). No significant difference in toxicity was noted between the two groups. Factors that were significantly associated with time to local tumor progression on multivariate analysis include prior WBRT (HR 1.62, p = 0.003), prior chemotherapy (HR 0.69, p = 0.011), margin dose (HR 0.88, p < 0.001) and radiographic features of melanin deposition (HR 3.73, p < 0.001), or clear hemorrhage (HR 2.20, p < 0.001). CONCLUSIONS: Our findings demonstrate that hemorrhagic intracranial melanoma metastases are associated with inferior local tumor control when treated with SRS, as compared to solid tumors. These results highlight the importance of early radiosurgery among patients with melanoma brain metastases before hemorrhage occurs.

Stereotactic Radiosurgery and Immune Checkpoint Inhibitors in the Management of Brain Metastases.

Brain metastases traditionally carried a poor prognosis with an overall survival of weeks to months in the absence of treatment. Radiation therapy modalities include whole brain radiation therapy (WBRT) and stereotactic radiosurgery (SRS). WBRT delivers a relatively low dose of radiation, has neurocognitive sequelae, and has not been investigated for its immunostimulatory effects. Furthermore, WBRT exposes the entire intracranial tumor immune microenvironment to radiation. SRS delivers a high dose of conformal radiation with image guidance to minimize dose to surrounding normal brain tissue, and appears to promote anti-tumor immunity. In parallel with many of these discoveries, immune checkpoint inhibitors (ICIs) have demonstrated a survival advantage in multiple malignancies commonly associated with brain metastases (e.g., melanoma). Combination SRS and ICI are theorized to be synergistic in anti-tumor immunity directed to brain metastases. The purpose of this review is to explore the synergy of SRS and ICIs, including pre-clinical data, existing clinical data, and ongoing prospective trials.

Quantitative proteomic profiling of bovine follicular fluid during follicle development.

Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy nonlactating Holstein cows to determine the protein expression profile of FF at key stages of follicular development. Follicles were individually aspirated in vivo at predeviation (F1 ∼ 7.0 mm), deviation (F1 ∼ 8.5 mm), postdeviation (F1 ∼ 12.0 mm), and preovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FFs from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled with TMTsixplex and quantified using liquid chromatography-mass spectrometry/mass spectrometry. A total of 143 proteins were identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P < 0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Among the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function.

Thermoregulatory responses of Holstein cows exposed to experimentally induced heat stress.

Heat stress (HS) adversely influences productivity and welfare of dairy cattle. We hypothesized that the thermoregulatory mechanisms vary depending on the exposure time to HS, with a cumulative effect on the adaptive responses and thermal strain of the cow. To identify the effect of HS on adaptive thermoregulatory mechanisms and predictors of caloric balance, Holstein cows were housed in climate chambers and randomly distributed into thermoneutral (TN; n=12) or HS (n=12) treatments for 16 days. Vaginal temperature (VT), rectal temperature (Tre), respiratory rate (RR), heart rate (HR), and dry matter intake (DMI) were measured. The temperature and humidity under TN were 25.9±0.2°C and 73.0±0.8%, respectively, and under HS were 36.3±0.3°C and 60.9±0.9%, respectively. The RR of the HS cows increased immediately after exposure to heat and was higher (76.02±1.70bpm, p<0.001) than in the TN (39.70±0.71bpm). An increase in Tre (39.87±0.07°C in the HS vs. 38.56±0.03°C in the TN, p<0.001) and in VT (39.82±0.10°C in the HS vs. 38.26±0.03°C in the TN, p<0.001) followed the increase in RR. A decrease (p<0.05) in HR occurred in the HS (62.13±0.99bpm) compared with the TN (66.23±0.79bpm); however, the magnitude of the differences was not the same over time. The DMI was lower in HS cows from the third day (8.27±0.33kgd-1 in the HS vs. 14.03±0.29kgd-1 in the TN, p<0.001), and the reduction of DMI was strongly affected (r=-0.65) by changes in the temperature humidity index. The effect of environmental variables from the previous day on physiological parameters and DMI was more important than the immediate effect, and ambient temperature represented the most determinant factor for heat exchange. The difference in the responses to acute and chronic exposure to HS suggests an adaptive response. Thus, intense thermal stress strongly influence thermoregulatory mechanisms and the acclimation process depend critically on heat exposure time.