Pathogenesis of Necrotizing Enterocolitis: Modeling the Innate Immune Response

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant''s intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice.Necrotizing enterocolitis (NEC) is a disorder characterized by intestinal necrosis in premature infants that results in significant morbidity and mortality.¹ Approximately 7% of infants with a birth weight between 500 and 1500 g develop NEC.¹ The pathogenesis is characterized by intestinal inflammation that can progress to systemic infection/inflammation, multiorgan failure, and death. The bowel is distended and hemorrhagic on gross inspection. On microscopic examination, signs of inflammation, mucosal edema, epithelial regeneration, bacterial overgrowth, submucosal gas bubbles, and ischemic transmural necrosis are seen (Figure 1, A–E).²Open in a separate windowFigure 1Examples of the various grades of morphological damage in hematoxylin and eosin–stained specimens. A–E: Representative samples of premature infants with necrotizing enterocolitis. A: Age-matched control from patient with jejunal atresia. B: Mild injury with hemorrhagic necrosis of mucosa and loss of villus tip architecture. C: Progressive injury with inflammatory infiltration of muscularis with complete villus destruction. D: Severe muscular and epithelial damage with complete loss of mucosa. E: Perforation with transmural necrosis with complete loss of epithelial and muscular architecture. F–J: Representative samples from intestinal injury secondary to gavage feeding in the setting of hypothermia and hypoxia in neonatal rats. F: Intact morphology, grade 0. G: Sloughing of villus tips, grade 1. H: Mid-villus necrosis, grade 2. I: Loss of villi, grade 3. J: Complete destruction of the mucosa, grade 4. Insets in F–J show higher magnified portions of the same sections, corresponding to the boxed regions. K–O: Representative images of tissue injury secondary to 60 minutes of intestinal ischemia and 90 minutes of reperfusion in 2-week-old mice. K: Sham-operated mice (no ischemia). L: Villus tip necrosis. M: Mid-villus necrosis. N: Loss of villus architecture. O: Complete loss of mucosal architecture. F–J, reprinted with permission from Nature Publishing Group.²⁸ Scale bars = 50 μm (A–E, K–O). Original magnification, ×20 (A–O, main images, and F–J, insets).Currently the pathogenesis of NEC is believed to have multifactorial causes, including intestinal immaturity and microbial dysbiosis. Intestinal immaturity leads to a compromised intestinal epithelial barrier, an underdeveloped immune defense, and altered vascular development and tone. The compromised epithelial barrier and underdeveloped immune system, when exposed to luminal microbiota that have been shaped by formula feedings, antibiotic exposure, and Cesarean delivery, can lead to intestinal inflammation and sepsis. Despite therapeutic success in animal model systems, there are relatively few therapeutic strategies that have allowed for significantly improved outcomes in infants with NEC. Two hurdles that persist are our incomplete understanding of the developing immune system in premature infants and our inability to adequately replicate these complex factors in animal models.^3,4 This review summarizes the complex intestinal immune system in premature infants and details what is known about the involvement of innate immune factors in NEC, both in animal models and in human disease.     

Beta1-integrin and IL-1alpha expression as bystander effect of medium from irradiated cells: the pilot study

Bystander effects have been proposed as a third action pathway of ionising radiation besides direct and indirect effects. The purpose of the study was to investigate whether expression of interleukin-1alpha (IL-1alpha) and beta1-integrin is elevated in bystander cells as a marker for bystander effects in comparison with classical markers such as the clonogenic assay, apoptosis and the presence of micronuclei. The hybrid cell line E.A. hy.926 obtained by fusion of HUVEC cells with the epithelial cell line A 459 was irradiated with 0-5 Gy. Bystander effects were established via medium transfer at 45 min and 4 h after irradiation from irradiated to nonirradiated cell populations. In order to exclude effects of the irradiated medium itself, irradiated medium only was also used for transfer to nonirradiated cells. Then, cells were fixed at 1, 2, 6, and 24 h after irradiation or medium transport and IL-1alpha and beta1-integrin were detected and evaluated. A higher number of beta1-integrin-positive cells was observed in both irradiated and bystander cell populations than in the control group at 1 and 24 h after irradiation with 1 Gy or medium transfer. Significantly higher numbers of IL-1alpha-positive cells were found at 1, 2, and 6 h after irradiation with 1 Gy or medium transfer as well as at 2 and 6 h after irradiation with 5 Gy or medium transfer. Clonogenic survival decreased dependently on the dose in irradiated cells but did not show any significant difference between the bystander cell populations and sham-irradiated cells. The irradiated medium itself did not have any effect. It is concluded that beta1-integrin and IL-1alpha expression may serve as more sensitive markers of post-irradiation responses in bystander cell populations than the classical radiobiological markers. Moreover, overexpression of beta1-integrin and IL-1alpha may induce increased susceptibility to inflammation of bystander cells.     

2-dimensional MEMS dielectrophoresis device for osteoblast cell stimulation

A fixed microelectrode device for cell stimulation has been designed and fabricated using micro-electro-mechanical systems (MEMS) technology. Dielectrophoretic forces obtained from non-uniform electric fields were used for manipulating and positioning osteoblasts. The experiments show that the osteoblasts experience positive dielectrophoresis (p-DEP) when suspended in iso-osmotic culture medium and exposed to AC fields at 5 MHz frequency. Negative dielectrophoresis (n-DEP) is obtained at 0.1 MHz. The viability of osteoblasts under dielectrophoresis has been investigated. The viability values for cells exposed to DEP are nearly three times higher than the control values, indicating that dielectrophoresis may have an anabolic effect on osteoblasts.     

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.     

SARS-CoV-2 Vaccine Responses in Individuals with Antibody Deficiency: Findings from the COV-AD Study

Background

Vaccination prevents severe morbidity and mortality from COVID-19 in the general population. The immunogenicity and efficacy of SARS-CoV-2 vaccines in patients with antibody deficiency is poorly understood.

Objectives

COVID-19 in patients with antibody deficiency (COV-AD) is a multi-site UK study that aims to determine the immune response to SARS-CoV-2 infection and vaccination in patients with primary or secondary antibody deficiency, a population that suffers from severe and recurrent infection and does not respond well to vaccination.

Methods

Individuals on immunoglobulin replacement therapy or with an IgG less than 4 g/L receiving antibiotic prophylaxis were recruited from April 2021. Serological and cellular responses were determined using ELISA, live-virus neutralisation and interferon gamma release assays. SARS-CoV-2 infection and clearance were determined by PCR from serial nasopharyngeal swabs.

Results

A total of 5.6% (n?=?320) of the cohort reported prior SARS-CoV-2 infection, but only 0.3% remained PCR positive on study entry. Seropositivity, following two doses of SARS-CoV-2 vaccination, was 54.8% (n?=?168) compared with 100% of healthy controls (n?=?205). The magnitude of the antibody response and its neutralising capacity were both significantly reduced compared to controls. Participants vaccinated with the Pfizer/BioNTech vaccine were more likely to be seropositive (65.7% vs. 48.0%, p?=?0.03) and have higher antibody levels compared with the AstraZeneca vaccine (IgGAM ratio 3.73 vs. 2.39, p?=?0.0003). T cell responses post vaccination was demonstrable in 46.2% of participants and were associated with better antibody responses but there was no difference between the two vaccines. Eleven vaccine-breakthrough infections have occurred to date, 10 of them in recipients of the AstraZeneca vaccine.

Conclusion

SARS-CoV-2 vaccines demonstrate reduced immunogenicity in patients with antibody deficiency with evidence of vaccine breakthrough infection.

     

Increased copy number at 17q22-q24 by CGH in breast cancer is due to high-level amplification of two separate regions

Studies by comparative genomic hybridization (CGH) have defined a chromosomal site at 17q22-q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types. Due to the limited resolution and dynamic range of CGH, it remains unclear whether this gain reflects high-level amplification of small subregion(s) or low-level gain of most of the distal 17q. We used 32 physically mapped 17q probes to construct more accurate copy number profiles for 14 breast cancer cell lines by interphase fluorescence in situ hybridization (FISH). Six cell lines (43%) showed an increased copy number of the 17q22-q24 region by CGH, and seven (50%) by FISH. FISH copy number profiles had a substantially higher dynamic range than did CGH profiles. FISH revealed two independent, highly amplified regions (A and B) at 17q23, separated by about 5 Mb of non-amplified DNA. These regions were distinctly telomeric from the ERBB2 gene locus. However, region A was often co-amplified with ERBB2, whereas B was amplified in cell lines that showed no ERBB2 amplification. We conclude that distal 17q gains recently discovered in breast cancer by CGH are due to high-level amplifications of two different regions at 17q23. This chromosomal region has previously been reported to undergo allelic loss and therefore was thought to harbor a tumor suppressor gene. The present FISH data provide support for the presence, and a starting point for the positional isolation, of 17q23 genes whose upregulation by amplification may play a role in the progression of breast cancer and many other tumor types. Genes Chromosomes Cancer 20:372–376, 1997. © 1997 Wiley-Liss, Inc.     

Artemether and tribendimidine lack activity in experimental treatment of Paragonimus westermani in the dog

Artemether and tribendimidine are active against several trematode species, but no data are available regarding the lung fluke Paragonimus westermani. We infected six dogs with 100 P. westermani metacercariae each. At day 103 post-infection, four dogs were treated orally for 3 days with either artemether (total dose, 66.7 and 75 mg/kg) or tribendimidine (total dose, 100 mg/kg). The remaining dogs were left untreated and served as control. Sixteen days after the final dosing, dogs were killed, and P. westermani flukes were recovered from the lungs and counted. Neither artemether nor tribendimidine showed activity against P. westermani at this dose regimen in dogs.     

Elastin point mutations cause an obstructive vascular disease, supravalvular aortic stenosis

Supravalvular aortic stenosis (SVAS) is an inherited obstructive vascular disease that affects the aorta, carotid, coronary and pulmonary arteries. Previous molecular genetic data have led to the hypothesis that SVAS results from mutations in the elastin gene, ELN. In these studies, the disease phenotype was linked to gross DNA rearrangements (35 and 85 kb deletions and a translocation) in three SVAS families. However, gross rearrangements of ELN have not been identified in most cases of autosomal dominant SVAS. To define the spectrum of ELN mutations responsible for this disorder, we refined the genomic structure of human ELN and used this information in mutational analyses. ELN point mutations co-segregate with the disease in four familial cases and are associated with SVAS in three sporadic cases. Two of the mutations are nonsense, one is a single base pair deletion and four are splice site mutations. In one sporadic case, the mutation arose de novo. These data demonstrate that point mutations of ELN cause autosomal dominant SVAS.