Different expression of caspase-3 in rat hippocampal subregions during postnatal development

Recent studies indicate that caspase-3 has distinct characteristics in postmitotic and neuronal progenitor apoptosis. Pyramidal neurons in CA1 and CA3 of the hippocampus become postmitotic during early postnatal development, whereas granule cells in the dentate gyrus (DG) undergo self-renewal throughout life. The distribution of caspase-3 in the hippocampal subfields during postnatal development is largely unknown. We used immunofluorescent staining for two isoforms of caspase-3 (an active 17 kDa isoform and an inactive 35 kDa precursor) and the Hoechst 33342 staining for nuclear chromatin to assess caspase-3 expression in the CA1, CA3, and DG of rat hippocampus during postnatal development. The expression of active caspase-3 reached a peak at P7 in CA1, at P2 in CA3, and then decreased with age. Whereas in DG, active caspase-3 expression increased slightly after P7, and remained at high levels for the rest of the investigated period. Procaspase-3 immunoreactivity was strong at P2 and decreased gradually to a basal plateau by P21 in the three regions examined. In addition, the number of apoptotic cells in the three regions all reached maximum levels at P7, and then decreased with age. These data indicate that there were specific spatio-temporal patterns of expression of active and precursor caspase-3 in the postnatally developing rat hippocampal subregions, and that the activation of caspase-3 in neuronal progenitor cells of DG and that in the postmitotic neurons of CA1 and CA3 may have distinct roles and mechanisms during postnatal development.     

Different expression of NR2B and PSD-95 in rat hippocampal subregions during postnatal development

The different expressions of NR2 and synaptic-associated proteins have been studied by protein and mRNA level with immunoblotting, in situ hybridization, or immunogold analysis. But the relationship between NR2 subunits and PSD-95 family proteins is still controversial. In this study, we used immunofluorescent staining to assess NR2B and PSD-95 expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on different postnatal day. In CA1, NR2B expression decreased with age. It was high at birth, reached a plateau at P4, and declined gradually after P7. In CA3, NR2B expression was similar to that in CA1. But the stratum lucidum was devoid of staining. In DG, the NR2B expression retained a higher level. From P0 to P2, the PSD-95 expression in CA1 increased gently, and then declined slightly. After P7, the PSD-95 expression increased sharply till P28, and decreased again. In CA3 and DG, the PSD-95 expression is very similar except that low-level of PSD-95 was found in the CA3 stratum lucidum. The expression of NR2B did not correlate with that of PSD-95 in CA1 and the DG granular and molecular layer. Only in CA3 and DG polymorphic layer, there was a negative correlation. The results suggested in hippocampal subregions, CA3 and DG may be more plastic than CA1.The NR2B and PSD-95 expression have distinct regional and cell specific distribution. The different regional distribution pattern may relate to the different physiological functions during postnatal development. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

8XC196单片机集成开发环境的研制

介绍用VB开发的单片机集成开发环境uBuilder软件。本软件的一个特色是具有源代码自动生成功能,它让用户逐个屏幕选择菜单、配置各种参数,然后模块就可以根据这些选项自动生成C语言或汇编语言源代码。用户可以直接将这些生成的代码拷贝到自己的应用程序中,这就大大减产了学习微控制器的时间和开发时间。     

Expression of TRPV4 in the zebrafish retina during development

The transient receptor potential (TRP) channels are involved in sensing mechanical/physical stimuli such as temperature, light, pressure, as well as chemical stimuli. Some TRP channels are present in the vertebrate retina, and the occurrence of the multifunctional channel TRP vanilloid 4 (TRPV4) has been reported in adult zebrafish. Here, we investigate the expression and distribution of TRPV4 in the retina of zebrafish during development using polymerase chain reaction (PCR), Western blot, and immunohistochemistry from 3 days post fertilization (dpf) until 100 dpf. TRPV4 was detected at the mRNA and protein levels in the eye of zebrafish at all ages sampled. Immunohistochemistry revealed the presence of TRPV4 in a population of the retinal cells identified as amacrine cells on the basis of their morphology and localization within the retina, as well as the co-localization of TRPV4 with calretinin. TRPV4 was first (3 dpf) found in the soma of cells localized in the inner nuclear and ganglion cell layers, and thereafter (10 dpf) also in the inner plexiform layer. The adult pattern of TRPV4 expression was achieved by 40 dpf the expression being restricted to the soma of some cells in the inner nuclear layer and ganglion cell layers. These data demonstrate the occurrence and developmental changes in the expression and localization of TRPV4 in the retina of zebrafish, and suggest a role of TRPV4 in the visual processing.     

Ultrastructural observations of programmed cell death during metanephric development in mouse

Previous studies revealed apoptosis as an only programmed cell death (PCD) during renal morphogenesis before alternative type of PCD, necroptosis were introduced. Evidences of non‐apoptotic PCD during renal development were scarce and needed to be accumulated. The purpose of this study is to investigate whether non‐apoptotic PCD is involved in and observe ultrastructural features of apoptotic cells or non‐apoptotic PCD during metanephros development. For this purpose, light and transmission electron microscopy were used. The most significant finding to come out of this study was that necroptosis was observed during developing metanephros by electron microscopy. The results also provided another fact that apoptosis and necroptosis constituted the PCD during embryonic development of kidney in mouse. Compared to necroptosis, apoptosis was more predominantly evident throughout whole development period and in every compartment of metanephros except for proximal tubule. However, necroptosis was only exhibited in developing nephrons also except for proximal tubule. In addition, outcomes of PCD were related to morphogenetic features of metanephric development. Efferocytosis for apoptotic cell or bodies took place in each type cell and whole period of developing metanephros. Besides efferocytosis blood flow and urine flux were available to remove the corpses of PCD, especially PCD from developing nephrons. Our findings suggested that both apoptosis and necroptosis play important roles during nephrogenesis and observed three ways to clear the PCD cell: efferocytosis, blood flow, and urine flux. Microsc. Res. Tech. 76:467–475, 2013. © 2013 Wiley Periodicals, Inc.     

Nitric oxide averts hypoxia-induced damage during reoxygenation in rat heart

Nitric oxide (NO), synthesized by the hemoproteins NO synthases (NOS), is known to play important roles in physiological and pathological conditions in the heart, including hypoxia/reoxygenation (H/R). This work investigates the role that endogenous NO plays in the cardiac H/R-induced injury. A follow-up study was conducted in Wistar rats subjected to 30 min of hypoxia, with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analysing parameters of cell, and tissue damage (lipid peroxidation, apoptosis, and protein nitration), as well as in situ NOS activity and NO production (NOx). The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated in all the experimental groups, and consequently, NOx levels fell. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. These results reveal that NOS inhibition exacerbates the peroxidative and apoptotic damage observed before the treatment with L-NAME in the hypoxic heart, pointing to a cardioprotective role of NOS-derived NO against H/R-induced injury. These findings could open the possibility of future studies to design new therapies for H/R-dysfunctions based on NO-pharmacology.     

Movement and behavior analysis using neural spike signals in CA1 of rat hippocampus

The hippocampus which lies in the temporal lobe plays an important role in spatial navigation,learning and memory.Several studies have been made on the place cell activity,spatial memory,prediction of future locations and various learning paradigms.However,there are no attempts which have focused on finding whether neurons which contribute largely to both spatial memory and learning about the reward exist.This paper proposes that there are neurons that can simultaneously engage in forming place memory and reward learning in a rat hippocampus' s CA1 area.With a trained rat,a reward experiment was conducted in a modified 8-shaped maze with five stages,and utterance information was obtained from a CA1 neuron.The firing rate which is the count of spikes per unit time was calculated.The decoding was conducted with log-maximum likelihood estimation(Log-MLE) using Gaussian distribution model.Our outcomes provide evidence of neurons which play a part in spatial memory and learning regarding reward.     

Involution dependent changes in distribution and localization of bax,survivin, caspase‐3, and calpain‐1 in the rat endometrium

The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis‐related proteins (bax and survivin) and enzymes (caspase‐3 and calpain‐1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase‐3, calpain‐1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain‐1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase‐3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non‐apoptotic activity of bax, caspase‐3, calpain‐1, and survivin. Microsc. Res. Tech. 79:285–297, 2016. © 2016 Wiley Periodicals, Inc.     

Impact of SARS-CoV-2 infection during pregnancy on postnatal brain development: The potential role of glial cells

Glial cells are crucial for maintaining central nervous system (CNS) homeostasis. They actively participate in immune responses, as well as form functional barriers, such as blood-brain barrier (BBB), which restrict the entry of pathogens and inflammatory mediators into the CNS. In general, viral infections during the gestational period can alter the embryonic and fetal environment, and the related inflammatory response may affect neurodevelopment and lead to behavioral dysfunction during later stage of life, as highlighted by our group for Zika virus infection. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induces a cytokine storm and, during pregnancy, may be related to a more severe form of the coronavirus disease-19 (COVID-19) and also to higher preterm birth rates. SARS-CoV-2 can also affect the CNS by inducing neurochemical remodeling in neural cells, which can compromise neuronal plasticity and synaptic function. However, the impact of SARS-CoV-2 infection during pregnancy on postnatal CNS, including brain development during childhood and adulthood, remains undetermined. Our group has recently highlighted the impact of COVID-19 on the expression of molecular markers associated with neuropsychiatric disorders, which are strongly related to the inflammatory response. Thus, based on these relationships, we discussed the impact of SARS-CoV-2 infection either during pregnancy or in critical periods of neurodevelopment as a risk factor for neurological consequences in the offspring later in life, focusing on the potential role of glial cells. Thus, it is important to consider future and long-term public health concerns associated with SARS-CoV-2 infection during pregnancy.     

基于ARM9的滚筒式生物芯片点样仪的研发

研发了一种新型滚筒式生物芯片点样仪,着重描述了点样仪的机械结构设计和控制系统软、硬件部分的设计.以基于ARM9 S3C2416微处理器带有触摸屏的控制器作为控制核心,实现了点样仪的双闭环高精度控制.最后用激光干涉仪测量了点样仪的控制精度,测试结果表明:系统定位精度为11 μm,重复精度为5μm,满足控制要求.现场实践证明:该点样仪结构设计合理、运行稳定、定位精准;硬件系统与软件系统巧妙结合,充分发挥了ARM9微处理器的硬件资源和触摸屏人机交互界面的强大功能,使点样仪控制系统具有可靠性高、实时性高、控制精度高等优点.     

The changes of gap junctions between pituitary folliculo‐stellate cells during the postnatal development of zucker fatty and lean rats

We investigated the effect of leptin on the postnatal development of gap junctions between folliculo‐stellate cells by using Zucker fatty (fa/fa) rats that have defects of the functional leptin receptor. Male Zucker fatty rats (fa/fa) and male Zucker lean rats (+/+) were used at each of the following postnatal ages: 20, 30, 40, 50, 60, 70, 80, 90 days, and 1 year. On one of the aforementioned dates, the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions, and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicular profile. In Zucker lean male rats, the number of gap junctions remained relatively constant from days 50 to 90 (0.44 ± 0.02 to 0.49 ± 0.03), and was similar in 1 year old rats (0.47 ± 0.03). These data were statistically higher compared to Zucker fatty male rats. In Zucker fatty male rats, very few gap junctions were observed in 30‐day‐old rats (0.04 ± 0.01: mean ± SE). This disruption of gap junction formation persisted, and the number of gap junctions remained constant and showed a low level from days 40 to 90 (0.11 ± 0.02 to 0.17 ± 0.02); this finding was similar in 1‐year‐old rats (0.17 ± 0.02). These observations indicate that the effect of leptin over the gap junction formation within the anterior pituitary glands was directly mediated by interaction with the functional leptin receptor present on the folliculo‐stellate cells. Microsc. Res. Tech. 77:31–36, 2014. © 2013 Wiley Periodicals, Inc.     

Accumulation of triosephosphate isomerase, with sequence homology to Beta amyloid peptides, in vessel walls of the newborn piglet hippocampus

We investigated whether beta-amyloid (Abeta)-like immunoreactivity was seen in the brains of newborn piglets. The immunoreactivity for Abeta(1-42) and Abeta(1-40) proteins, but not Abeta precursor protein, was present in CD68-positive perivascular cells of the hippocampus and in parts of the meninges. It was colocalized with immunoreactivity for receptor for advanced glycation end product and tumor necrosis factor-alpha. The protein with a molecular mass of 27 kDa, which was recognized by the Abeta antibodies, was identified as triosephosphate isomerase (TPI) with sequence homology to Abeta peptides by N-terminal amino acid sequencing, mass fingerprint analysis using matrix-associated laser desorption/ionization mass spectrometry, and Western blotting. Western blotting assay also revealed that detectable expression of Abeta proteins were not seen in the piglet brains. These findings indicate that TPI with sequence homology to Abeta peptides accumulates in perivascular cells of the microglia/macrophage lineage located around arterial vessels of the newborn piglet hippocampus.     

Modified cuckoo search algorithm in microscopic image segmentation of hippocampus

Microscopic image analysis is one of the challenging tasks due to the presence of weak correlation and different segments of interest that may lead to ambiguity. It is also valuable in foremost meadows of technology and medicine. Identification and counting of cells play a vital role in features extraction to diagnose particular diseases precisely. Different segments should be identified accurately in order to identify and to count cells in a microscope image. Consequently, in the current work, a novel method for cell segmentation and identification has been proposed that incorporated marking cells. Thus, a novel method based on cuckoo search after pre‐processing step is employed. The method is developed and evaluated on light microscope images of rats’ hippocampus which used as a sample for the brain cells. The proposed method can be applied on the color images directly. The proposed approach incorporates the McCulloch's method for lévy flight production in cuckoo search (CS) algorithm. Several objective functions, namely Otsu's method, Kapur entropy and Tsallis entropy are used for segmentation. In the cuckoo search process, the Otsu's between class variance, Kapur's entropy and Tsallis entropy are employed as the objective functions to be optimized. Experimental results are validated by different metrics, namely the peak signal to noise ratio (PSNR), mean square error, feature similarity index and CPU running time for all the test cases. The experimental results established that the Kapur's entropy segmentation method based on the modified CS required the least computational time compared to Otsu's between‐class variance segmentation method and the Tsallis entropy segmentation method. Nevertheless, Tsallis entropy method with optimized multi‐threshold levels achieved superior performance compared to the other two segmentation methods in terms of the PSNR.     

Cell death and differentiation in the development of the endocardial cushion of the embryonic heart

The transformation of the endocardial cushion into valves and septa is a critical step in cardiac morphogenesis as it initiates the development of the four-chambered heart. This transformation results from a region-specific balance between cellular proliferation, apoptosis, and differentiation. The development of the form and structure of the endocardial cushion is accompanied by precise patterns of abundant cell death having the morphological features of programmed cell death (apoptosis), which plays an important role in the elimination of redundant cells and in changes of phenotypic composition during histogenesis. Apoptosis is an essential process in morphogenesis as it balances mitosis in renewing tissues. It is controlled by one or more genetic programs that kill the targeted cell. However, the causes, role, and regulation of apoptosis in the developing endocardial cushion still remain to be determined. The clarification of the role of the apoptosis regulatory genes constitutes a major task in future studies of cell death in the developing heart. This new molecular histology of heart development awaits further experiments to clarify the interactive mechanisms that act to ensure the sculpting of the endocardial cushion into valves and septa by determining the size of the cushion cell populations. The relation between the expression of different factors and the modifications of the cushion region during cardiac development are reviewed. In addition, we review and summarize information on molecules identified in our experiments that imply the activity of a number of essential genes coinciding with the key steps in generating the overall architecture of the heart. We correlate their temporal and spatial expression with their proposed roles.     

Immunohistological evidences of Ginkgo biloba extract altering Bax to Bcl-2 expression ratio in the hippocampus and motor cortex of senescence accelerated mice

Ginkgo biloba extract 761 appears to display neuroprotective effect in many nervous diseases and aging. Deterioration of mental functions during aging is always accompanied by loss of neurons, presumably through apoptosis. Here, we studied the effect of G. biloba extract in the expression of Bax/Bcl-2 ratio, an important apoptotic index, in the hippocampus and motor cortex of the aging brain. Bax and Bcl-2 expressions were examined with immunohistochemical methods. Senescence Accelerated Mice Prone Strain 8 was used because the aging process was accelerated with neuropathological alterations similar to those found in the aging human brain. The mice were fed with either G. biloba extract or sucrose from the age of 3 weeks until sacrifice at 3 or 9 months old. In the hippocampus of G. biloba fed 9-month-old mice, the ratio of Bax positive cell to Bcl-2 positive cell (Bax/Bcl-2 expression ratio) was 11.43 +/- 3.11 (mean +/- SD); significantly lower (P < 0.05) than the Bax/Bcl-2 expression ratio of 20.99 +/- 5.34 in the sucrose fed mice. The Bax/Bcl-2 expression cell ratios, however, in the motor cortex were not significantly different between the two groups (2.22 +/- 1.35 versus 2.27 +/- 2.02 for the G. biloba and the sucrose fed mice, respectively). The decrease in the Bax/Bcl-2 expression cell ratio following G. biloba treatment might hence be able to protect the aging hippocampus from moving further down the apoptotic pathway. Western blotting confirmed the decrease of Bax in the brain even after a short term and high dose Ginkgo treatment. It is speculated that the G. biloba extract may be a potential neuroprotective agent against apoptosis through the differential expressions of the Bax and Bcl-2 in the hippocampus.     

Expression and localization of translationally controlled tumor protein in rat urinary organs

This study describes the very first assessment of the expression and localization of translationally controlled tumor protein (TCTP) in adult rat urinary organs. TCTP expression levels in kidneys, urinary bladder, and urethra were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting, and its cellular localization was examined immunohistochemically in paraffin sections of various urinary organs. TCTP was found in all urinary organs. Its expression was high in the urethra and low in the bladder. TCTP was localized in glomerular podocytes, epithelium of proximal and distal renal tubules, in the loop of Henle, and in the transitional epithelium of the bladder and urethra, mostly in basal cell layers). The subcellular localization of TCTP in these urinary organs was cytoplasmic. These findings suggest that TCTP may be involved in urine formation and excretion. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Nuclear pores in luteal cells during pregnancy and after parturition and pup removal in the rat. A freeze-fracture study

In a previous paper we described a pronounced increase of apoptotic nuclei in rat corpus luteum of pregnancy whose programmed chromatin degeneration was induced by the progesterone antagonist mifepristone. Those observations encouraged us to study the apoptotic nuclear membrane during pregnancy and after parturition and pup removal, by using a freeze-fracture technique which allows us to observe ‘en face’ the nuclear envelop and also permits nuclear pore counting. This study was complemented with the TUNEL assay (TdT-mediated dUTP nick-end labelling). Changes in nuclear pores during pregnancy begin with an intense reduction in number but still showing an even distribution on the nuclear membrane, never forming aggregations sharply separated from pore-free areas, which are characteristic of other apoptotic models. Electron microscopy of thin-sections shows, coincidently with findings in the freeze-fracture replicas, a moderately irregular aggregation of marginal heterochromatin condensations. After nuclear fragmentation and micronuclear formation, pores behave in the usual manner in other apoptotic models, i.e., mainly showing migrations of nuclear pores toward the chromatin-free areas. The present results support the hypothesis that nuclear pore complexes are dynamic structures, which permit their migration toward nuclear membrane areas devoid of chromatin aggregations that might block the nucleocytoplasmic transport in such areas.     

The administration of nonmetabolizable glucose analogues fails to suppress the development of glycogen autophagy in newborn rat hepatocytes

The effects of parenteral administration of glucose, 3‐methylglucose (3MG), or 2‐deoxyglucose (2DG) on the glycogen autophagy were studied in the newborn rat liver using electron microscopy and biochemical methods. The administration of glucose resulted in hyperglycemia and prevented the mobilization of hepatocytic glycogen. It also prevented the development of autophagic vacuoles in general and inhibited the glycogen‐degrading activity of acid α‐1,4‐glucosidase. The nonphosphorylated and not further metabolized glucose analog 3MG also produced hyperglycemia, but increased acid glucosidase. Pretreating the newborns with the β‐adrenergic blocker propranolol inhibited the effects of 3MG. The phosphorylated but not fully metabolized glucose analog 2DG produced similar effects. The administration of xylitol to the newborns already treated with 2DG, suppressed acid glucosidase. The results of this and our previous studies suggest that glucose must be metabolized beyond its phosphorylation step to inhibit acid glucosidase activity. Microsc. Res. Tech. 73:1009–1014, 2010. © 2010 Wiley‐Liss, Inc.     

Drift study of SU8 cantilevers in liquid and gaseous environments

We present a study of the drift, in terms of cantilever deflections without probe/target interactions, of polymeric SU8 cantilevers. The drift is measured in PBS buffer (pH 7.4) and under vacuum (1 mbar) conditions. We see that the cantilevers display a large drift in both environments. We believe this is because the polymer matrix absorbs liquid in one situation whereas it is being degassed in the other. An inhomogeneous expansion/contraction of the cantilever is seen because one surface of the cantilever may still have remains of the release layer from the fabrication. To further study the effect, we coat the cantilevers with a hydrophobic coating, perfluorodecyltrichlorosilane (FDTS). Fully encapsulating the SU8 cantilever greatly reduces the drift in liquid whereas a less significant change is seen in vacuum.     

Identification of differential mRNA and lncRNA expression in AcMNPV-infected Sf9 cells

Sf9Sf9 are the ovarian cells of Spodoptera frugiperda that is the host of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and hence can serve as an effective test vehicle to understand the AcMNPV infection mechanism. In this study, through high-throughput sequencing technology using samples collected from Sf9 cells at different time points after AcMNPV infection, 3463 pieces of time-series differentially expressed RNA (1,200 mRNA and 2,263 lncRNA) are identified and justified by experimental verification of randomly selected samples from them, proving the validity of the bioinformatical analysis on this topic. Functional enrichment analysis and target prediction are performed on those differentially expressed RNA, from which the major functional enrichment distribution of those differentially expressed mRNA is derived. It has been found that the differential genes are mainly in the cellular anatomical entity and intracellular in terms of the cellular component, and in the binding and catalytic activity in terms of the molecular function. Also, the differential mRNA are mainly concentrated in global and overview maps, signal transduction, infectious diseases, and viral, etc. Moreover, those mRNA targeted by lncRNA are predicted. The correlation between those differentially expressed lncRNA and mRNA indicates that lncRNA is very likely playing an important role in the interaction between virus and host. Aided by an advanced co-expression analysis approach, the “hub” RNA is also identified. The study in this work pave the way for further analyzing and understanding how AcMNPV escapes from the host’s immunity, manipulates the host to realize the self-multiplication, and realizes the timely conversion between its two particle forms, laying the foundation for uncovering the host’s immune response process.