Molecular functions of ANKLE2 and its implications in human disease.

Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.

A Classification-Based Adaptive Segmentation Pipeline: Feasibility Study Using Polycystic Liver Disease and Metastases from Colorectal Cancer CT Images.

Automated segmentation tools often encounter accuracy and adaptability issues when applied to images of different pathology. The purpose of this study is to explore the feasibility of building a workflow to efficiently route images to specifically trained segmentation models. By implementing a deep learning classifier to automatically classify the images and route them to appropriate segmentation models, we hope that our workflow can segment the images with different pathology accurately. The data we used in this study are 350 CT images from patients affected by polycystic liver disease and 350 CT images from patients presenting with liver metastases from colorectal cancer. All images had the liver manually segmented by trained imaging analysts. Our proposed adaptive segmentation workflow achieved a statistically significant improvement for the task of total liver segmentation compared to the generic single-segmentation model (non-parametric Wilcoxon signed rank test, n = 100, p-value << 0.001). This approach is applicable in a wide range of scenarios and should prove useful in clinical implementations of segmentation pipelines.

Chemokine expression profile of an innate granuloma.

Granulomas are defined by the presence of organized layers of immune cells that include macrophages. Granulomas are often characterized as a way for the immune system to contain an infection and prevent its dissemination. We recently established a mouse infection model where Chromobacterium violaceum induces the innate immune system to form granulomas in the liver. This response successfully eradicates the bacteria and returns the liver to homeostasis. Here, we sought to characterize the chemokines involved in directing immune cells to form the distinct layers of a granuloma. We use spatial transcriptomics to investigate the spatial and temporal expression of all CC and CXC chemokines and their receptors within this granuloma response. The expression profiles change dynamically over space and time as the granuloma matures and then resolves. To investigate the importance of monocyte-derived macrophages in this immune response, we studied the role of CCR2 during C. violaceum infection. Ccr2 -/- mice had negligible numbers of macrophages, but large numbers of neutrophils, in the C. violaceum-infected lesions. In addition, lesions had abnormal architecture resulting in loss of bacterial containment. Without CCR2, bacteria disseminated and the mice succumbed to the infection. This indicates that macrophages are critical to form a successful innate granuloma in response to C. violaceum.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

Precise control of protein ubiquitination is essential for brain development, and hence, disruption of ubiquitin signaling networks can lead to neurological disorders. Mutations of the deubiquitinase USP7 cause the Hao-Fountain syndrome (HAFOUS), characterized by developmental delay, intellectual disability, autism, and aggressive behavior. Here, we report that conditional deletion of USP7 in excitatory neurons in the mouse forebrain triggers diverse phenotypes including sensorimotor deficits, learning and memory impairment, and aggressive behavior, resembling clinical features of HAFOUS. USP7 deletion induces neuronal apoptosis in a manner dependent of the tumor suppressor p53. However, most behavioral abnormalities in USP7 conditional mice persist despite p53 loss. Strikingly, USP7 deletion in the brain perturbs the synaptic proteome and dendritic spine morphogenesis independently of p53. Integrated proteomics analysis reveals that the neuronal USP7 interactome is enriched for proteins implicated in neurodevelopmental disorders and specifically identifies the RNA splicing factor Ppil4 as a novel neuronal substrate of USP7. Knockdown of Ppil4 in cortical neurons impairs dendritic spine morphogenesis, phenocopying the effect of USP7 loss on dendritic spines. These findings reveal a novel USP7-Ppil4 ubiquitin signaling link that regulates neuronal connectivity in the developing brain, with implications for our understanding of the pathogenesis of HAFOUS and other neurodevelopmental disorders.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover an innate granuloma model in mice with an environmental bacterium called Chromobacterium violaceum. Granuloma formation not only successfully walls off, but also clears, the infection. The infected lesion can arise from a single bacterium that replicates despite the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum-induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. This innate granuloma successfully eradicates C. violaceum infection. Therefore, this C. violaceum-induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby resolve infection by an environmental pathogen.

What Can Groundwater Monitoring Tell Us About Gas Migration? A Numerical Modeling Study.

Groundwater monitoring to measure a variety of indicator parameters including dissolved gas concentrations, total dissolved gas pressure (TDGP), and redox indicators is commonly used to evaluate the impacts of gas migration (GM) from energy development in shallow aquifer systems. However, these parameters can be challenging to interpret due to complex free-phase gas source architecture, multicomponent partitioning, and biogeochemical reactions. A series of numerical simulations using a gas flow model and a reactive transport model were conducted to delineate the anticipated evolution of indicator parameters following GM in an aquifer under a variety of physical and biogeochemical conditions. The simulations illustrate how multicomponent mass transfer processes and biogeochemical reactions create unexpected spatial and temporal variations in several analytes. The results indicate that care must be taken when interpreting measured indicator parameters including dissolved hydrocarbon concentrations and TDGP, as the presence of dissolved gases in background groundwater and biogeochemical processes can cause potentially misleading conclusions about the impact of GM. Based on the consideration of multicomponent gas partitioning in this study, it is suggested that dissolved background gases such as N2 and Ar can provide valuable insights on the presence, longevity and fate of free-phase natural gas in aquifer systems. Overall, these results contribute to developing a better understanding of indicators for GM in groundwater, which will aid the planning of future monitoring networks and subsequent data interpretation.

Humoral Response to the Acetalated Dextran M2e Vaccine is Enhanced by Antigen Surface Conjugation.

The influenza A virus causes substantial morbidity and mortality worldwide every year and poses a constant threat of an emergent pandemic. Seasonal influenza vaccination strategies fail to provide complete protection against infection due to antigenic drift and shift. A universal vaccine targeting a conserved influenza epitope could substantially improve current vaccination strategies. The ectodomain of the matrix 2 protein (M2e) of influenza is a highly conserved epitope between virus strains but is also poorly immunogenic. Administration of M2e and the immunostimulatory stimulator of interferon genes (STING) agonist 3'3'-cyclic guanosine-adenosine monophosphate (cGAMP) encapsulated in microparticles made of acetalated dextran (Ace-DEX) has previously been shown to be effective for increasing the immunogenicity of M2e, primarily through T-cell-mediated responses. Here, the immunogenicity of Ace-DEX MPs delivering M2e was further improved by conjugating the M2e peptide to the particle surface in an effort to affect B-cell responses more directly. Conjugated or encapsulated M2e co-administered with Ace-DEX MPs containing cGAMP were used to vaccinate mice, and it was shown that two or three vaccinations could fully protect against a lethal influenza challenge, while only the surface-conjugated antigen constructs could provide some protection against lethal challenge with only one vaccination. Additionally, the use of a reducible linker augmented the T-cell response to the antigen. These results show the utility of conjugating M2e to the surface of a particle carrier to increase its immunogenicity for use as the antigen in a universal influenza vaccine.

Exophytic Squamous Cell Carcinoma of the Scalp: A Case Report.

Squamous cell carcinoma (SCC) is the second-most common clinical presentation of non-melanoma skin cancer. Despite its prevalence, the rate of growth and development of SCC lesions is low. We present a case report of an exophytic SCC of the midline scalp. Over approximately 18 months, the exophytic portion of this lesion grew to a size of 8.5 x 7 cm due to the fact that the patient did not seek medical attention. The patient suffered from many predisposing factors including active smoking status, type II diabetes, and significant previous sun exposure. In addition to these predisposing factors, the patient did not have comprehensive health insurance to cover outpatient medical care. This case highlights the importance of early intervention in the management of head and neck skin cancers and the negative impact of delayed treatment.

Primary Repair of Peroneus Longus Myofascial Herniation With Symptomatic Superficial Peroneal Nerve Compression.

Symptomatic myofascial herniations of the extremities occur infrequently; however, they can contribute to significant pain, weakness, and neuropathy with activity. Muscle herniation typically occurs through either a traumatic or congenital focal defect in the deep overlying fascia. Patients may present with an intermittently palpable subcutaneous mass and may have neuropathic symptoms, depending on the degree of nerve involvement. Patients are initially treated with conservative modalities, whereas surgery is reserved for patients who demonstrate persistent functional limitations and neurologic symptoms. Here, we demonstrate a technique for primary repair of a symptomatic lower-leg fascial defect.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover a novel granuloma model in mice. Chromobacterium violaceum is an environmental bacterium that stimulates granuloma formation that not only successfully walls off but also clears the infection. The infected lesion can arise from a single bacterium that replicates in the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum -induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. These innate granulomas successfully eradicate C. violaceum infection. Therefore, this new C. violaceum -induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby eradicate infection by an environmental pathogen.

Ectopic Fibrous Dysplasia Presenting As a Well-Circumscribed Orbital Mass.

The authors present the case of a middle-aged woman with subacute progressive swelling of the OD associated with pain and severely limited duction in all gazes. MRI demonstrated a ~3 cm circumscribed mass in the extraconal space, which displaced and distorted the globe and impinged upon ocular adnexa. She underwent lateral orbitotomy with bone osteotomy to remove the mass, and subsequent histopathologic examination showed woven bone set within a fibrotic background, microscopic features of fibrous dysplasia. Fibrous dysplasia is characterized by abnormal scar-like bony proliferation, typically within a preexisting structure of the skeletal system. To the authors' knowledge, this represents the first example of fibrous dysplasia presenting as an orbital mass unconnected to the craniofacial skeleton.

Use of High-Resolution Ultrasound to Guide Alcohol Neurolysis for Chronic Pain.

BACKGROUND: The diagnosis and treatment of neuropathic pain is often clinically challenging, with many patients requiring treatments beyond oral medications. To improve our percutaneous treatments, we established a clinical pathway that utilized ultrasound (US) guidance for steroid injection and alcohol ablation for patients with painful neuropathy. OBJECTIVES: To describe a collaborative neuropathy treatment pathway developed by a neurosurgeon, pain physicians, and a sonologist, describing early clinical experiences and patient-reported outcomes. STUDY DESIGN: A retrospective case series was performed. METHODS: Patients that received percutaneous alcohol ablation with US guidance for neuropathy were identified through a retrospective review of a single provider's case log. Demographics and treatment information were collected from the electronic medical record. Patients were surveyed about their symptoms and treatment efficacy. Descriptive statistics were expressed as medians and the interquartile range ([IQR]; 25th and 75th data percentiles). Differences in the median follow-up pain scores were assessed using a Wilcoxon signed-rank test. RESULTS: Thirty-five patients underwent US-guided alcohol ablation, with the average patient receiving one treatment (range: 1 to 2), having a median duration of 4.8 months until reinjection (IQR: 2.9 to 13.1). The median number of steroid injections that individuals received before US-guided alcohol ablation was 2 (IQR: 1 to 3), and the median interval between steroid injections was 3.7 months (IQR: 2.0 to 9.6). Most (20/35 [57%]) patients responded to the survey, and the median pain scores decreased by 3 units (median: -3, IQR: -6 to 0; P < 0.001) one week following the alcohol ablation. This pain reduction remained significant at one month (P < 0.001) and one year (P = 0.002) following ablation. Most (12/20 [60%]) patients reported that alcohol ablation was more effective in improving their pain than oral pain medications. LIMITATIONS: Given the small sample size, treatment efficacy for alcohol neurolysis cannot be generalized to the broader population. CONCLUSIONS: US-guided percutaneous treatments for neuropathic pain present a growing opportunity for interprofessional collaboration between neurosurgery, clinicians who treat chronic pain, and sonologists. US can provide valuable diagnostic information and guide accurate percutaneous treatments in skilled hands. Further studies are warranted to determine whether a US-guided treatment pathway can prevent unnecessary open surgical management.

Microparticle Delivery of a STING Agonist Enables Indirect Activation of NK Cells by Antigen-Presenting Cells.

Natural killer (NK) cells are an important member of the innate immune system and can participate in direct tumor cell killing in response to immunotherapies. One class of immunotherapy is stimulator of interferon gene (STING) agonists, which result in a robust type I interferon (IFN-I) response. Most mechanistic studies involving STING have focused on macrophages and T cells. Nevertheless, NK cells are also activated by IFN-I, but the effect of STING activation on NK cells remains to be adequately investigated. We show that both direct treatment with soluble STING agonist cyclic di-guanosine monophosphate-adenosine monophosphate (cGAMP) and indirect treatment with cGAMP encapsulated in microparticles (MPs) result in NK cell activation in vitro, although the former requires 100× more cGAMP than the latter. Additionally, direct activation with cGAMP leads to NK cell death. Indirect activation with cGAMP MPs does not result in NK cell death but rather cell activation and cell killing in vitro. In vivo, treatment with soluble cGAMP and cGAMP MPs both cause short-term activation, whereas only cGAMP MP treatment produces long-term changes in NK cell activation markers. Thus, this work indicates that treatment with an encapsulated STING agonist activates NK cells more efficiently than that with soluble cGAMP. In both the in vitro and in vivo systems, the MP delivery system results in more robust effects at a greatly reduced dosage. These results have potential applications in aiding the improvement of cancer immunotherapies.

The Epidemiology and Outcome of Pericardial Effusion in Hospitalized Children: A National Database Analysis.

OBJECTIVES: To describe the epidemiology of pericardial effusion in hospitalized children and evaluate risk factors associated with the drainage of pericardial effusion and hospital mortality. STUDY DESIGN: A retrospective study of a national pediatric discharge database. RESULTS: We analyzed hospitalized pediatric patients from the neonatal age through 20 years in the Kids' Inpatient Database 2016, extracting the cases of pericardial effusion. Of the 6 266 285 discharged patients recorded, 6417 (0.1%) were diagnosed with pericardial effusion, with the highest prevalence of 2153 patients in teens (13-20 years of age). Pericardial effusion was drained in 792 (12.3%), and the adjusted risk of pericardial drainage was statistically low with rheumatologic diagnosis (OR, 0.485; 95% CI, 0.358-0.657, P < .001). The overall mortality in children with pericardial effusion was 6.8% and 10.9% of those who required pericardial effusion drainage (P < .001). The adjusted risk of mortality was statistically high with solid organ tumor (OR, 1.538; 95% CI, 1.056-2.239, P = .025) and pericardial drainage (OR, 1.430; 95% CI, 1.067-1.915, P = .017) and low in all other age groups compared with neonates, those with cardiac structural diagnosis (OR, 0.322; 95% CI, 0.212-0.489, P < .001), and those with rheumatologic diagnosis (OR, 0.531; 95% CI, 0.334-0.846, P = .008). CONCLUSION: The risk of mortality in hospitalized children with pericardial effusion was higher in younger children with solid organ tumors and those who required pericardial effusion drainage. In contrast, it was lower in older children with cardiac or rheumatologic diagnoses.

Development of a convolutional neural network to detect abdominal aortic aneurysms.

Objective: We sought to train a foundational convolutional neural network (CNN) for screening computed tomography (CT) angiography (CTA) scans for the presence of infrarenal abdominal aortic aneurysms (AAAs) for future predictive modeling and other artificial intelligence applications. Methods: From January 2015 to January 2020, a HIPAA (Health Insurance and Accountability Act)-compliant, institutional review board-approved, retrospective clinical study analyzed contrast-enhanced abdominopelvic CTA scans from 200 patients with infrarenal AAAs and 200 propensity-matched control patients with non-aneurysmal infrarenal abdominal aortas. A CNN was trained to binary classification on the input. For model improvement and testing, transfer learning using the ImageNet database was applied to the VGG-16 base model. The image dataset was randomized to sets of 60%, 10%, and 30% for model training, validation, and testing, respectively. A stochastic gradient descent was used for optimization. The models were assessed by testing validation accuracy and the area under the receiver operating characteristic curve. Results: Preliminary data demonstrated a nonrandom pattern of accuracy and detectability. Iterations (≤10) of the model characteristics generated a final custom CNN model reporting an accuracy of 99.1% and area under the receiver operating characteristic curve of 0.99. Misjudgments were analyzed through review of the heat maps generated via gradient weighted class activation mapping overlaid on the original CT images. The greatest misjudgments were seen in small aneurysms (<3.3 cm) with mural thrombus. Conclusions: Preliminary data from a CNN model have shown that the model can accurately screen and identify CTA findings of infrarenal AAAs. This model serves as a proof-of-concept to proceed with potential future directions to include expansion to predictive modeling and other artificial intelligence-based applications.

Multiplexed electrospray enables high throughput production of cGAMP microparticles to serve as an adjuvant for a broadly acting influenza vaccine.

Subunit vaccines employing designer antigens such as Computationally Optimized Broadly Reactive Antigen (COBRA) hemagglutinin (HA) hold the potential to direct the immune response toward more effective and broadly-neutralizing targets on the Influenza virus. However, subunit vaccines generally require coadministration with an adjuvant to elicit a robust immune response. One such adjuvant is the stimulator of interferon genes (STING) agonist cyclic dinucleotide 3'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). We have shown that encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles through electrospray results in significantly greater biological activity. Electrospray is a continuous manufacturing process which achieves excellent encapsulation efficiency. However, the throughput of electrospray with a single spray head is limited. Here we report the development of a multiplexed electrospray apparatus with an order of magnitude greater throughput than a single-head apparatus. Physicochemical characterization and evaluation of adjuvant activity in vitro and in vivo indicated that microparticles produced with the higher throughput process are equally suited for use as a potent vaccine adjuvant to induce a balanced immune response to COBRA HA antigens.

The nature of gas production patterns associated with methanol degradation in natural aquifer sediments: A microcosm study.

With growing global use of methanol as a fuel additive and extensive use in other industrial processes, there is the potential for unintended release and spills into soils and aquifers. In these subsurface systems it is likely that methanol will be readily biodegraded; however, degradation may lead to the production of by-products, most importantly methane possibly resulting in explosion hazards and volatile fatty acids (VFAs) causing aesthetic issues for groundwater. In this study, the formation of these potentially harmful by-products due to methanol biodegradation was investigated in natural sand and silt sediments using microcosms inoculated with neat methanol (100%) ranging in concentration from 100 to 100,000 ppm. To assess the rate of degradation and by-product formation, water and headspace samples were collected and analyzed for methanol, volatile fatty acids (VFAs, including acetic, butyric, and propionic acid), cation (metal) concentrations (Al, Ca, Fe, K, Mg, Mn and Na), microbial community structure and activity, headspace pressure, gas composition (CH4, CO2, O2 and N2), and compound specific isotopes. Methanol was completely biodegraded in sand and silt up to concentrations of 1000 ppm and 10,000 ppm, respectively. Degradation was initially aerobic, consuming oxygen (O2) and producing carbon dioxide (CO2). When O2 was depleted, the microcosms became anaerobic and a lag in methanol degradation occurred (ranging from 41 to 87 days). Following this lag, methanol was preferentially degraded to acetate, coupled with CO2 reduction. Microcosms with high methanol concentrations (10,000 ppm) were driven further down the redox ladder and exhibited fermentation, leading to concurrent acetate and methane (CH4) generation. In all cases acetate was an intermediate product, further degraded to the final products of CH4 and CO2. Carbonates present in the microcosm sediments helped buffer VFA acidification and replenished CO2. Methane generation in the anaerobic microcosms was short-lived, but temporarily reached high rates up to 13 mg kg-1 day-1. Under the conditions of these experiments, methanol degradation occurred rapidly, after initial lag periods, which were a function of methanol concentration and sediment type. Our experiment also showed that methanol degradation and associated methane production can occur in a stepwise fashion.

Development of an Intranasal Gel for the Delivery of a Broadly Acting Subunit Influenza Vaccine.

Influenza virus is a major cause of death on a global scale. Seasonal vaccines have been developed to combat influenza; however, they are not always highly effective. One strategy to develop a more broadly active influenza vaccine is the use of multiple rounds of layered consensus buildings to generate recombinant antigens, termed computationally optimized broadly reactive antigen (COBRA). Immunization with the COBRA hemagglutinin (HA) can elicit broad protection against multiple strains of a single influenza subtype (e.g., H1N1). We formulated a COBRA H1 HA with a stimulator of interferon genes agonist cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) into a nasal gel for vaccination against influenza. The gel formulation was designed to increase mucoadhesion and nasal retention of the antigen and adjuvant to promote a strong mucosal response. It consisted of a Schiff base-crosslinked hydrogel between branched polyethyleneimine and oxidized dextran. Following a prime-boost-boost schedule, an intranasal gel containing cGAMP and model antigen ovalbumin (OVA) led to the faster generation of serum IgG, IgG1, and IgG2c and significantly greater serum IgG1 levels on day 42 compared to soluble controls. Additionally, OVA-specific IgA was detected in nasal, vaginal, and fecal samples for all groups, except the vehicle control. When the COBRA HA was given intranasally in a prime-boost schedule, the mice receiving the gel containing the COBRA and cGAMP had significantly higher serum IgG and IgG2c at day 41 compared to all groups, and only this group had IgA levels above the background in vaginal, nasal, and fecal samples. Overall, this study indicates the utility of an intranasal gel for the delivery of COBRAs for the generation of serum and mucosal humoral responses.