Disorganization Of Endoplasmic Reticulum Research Articles

Long-term arsenic exposure decreases mice body weight and liver lipid droplets

Arsenic (As) is a widespread global pollutant, and there is significant controversy surrounding its complex relationship with obesity, primarily focused on short-term exposure. Recognizing the prolonged nature of dietary arsenic exposure, this study involved feeding mice with arsenic-contained food for 14 months. The results showed that mice exposed to arsenic developed a non-alcoholic fatty liver condition, characterized by a light-yellow hue on the liver surface and various pathological alterations in the liver cells, including enlarged nuclei, cellular necrosis, inflammatory infiltration, dysfunctional mitochondria, and endoplasmic reticulum disorganization. There were also disruptions in biochemistry indices, with a significant increase in total cholesterol (TC) level and a decrease in high-density lipoprotein (HDL) level. However, some contradictory observations occurred, such as a significant decrease in body weight, triglyceride (TG) level, and the numbers of lipid droplets. Several genes related to lipid metabolism were tested, and a model was used to explain these discrepancies. Besides, examinations of the colon revealed compromised intestinal barrier function and signs of inflammation. Fecal 16S rRNA sequencing and pseudo-targeted metabolomics revealed disruptions in internal homeostasis, such as modules, nodes, connections, and lipid-related KEGG pathways. Fecal targeted metabolomics analyses of short-chain fatty acids (SCFAs) and bile acids (BAs) demonstrated a significant upregulation in three primary bile acids (CA, CDCA, TCDCA), four secondary bile acids (TUDCA, DCA, LCA, GUDCA), and total SCFAs level. Oxidative stress and inflammation were also evident. Additionally, based on correlation analysis and mediation analysis, it was assumed that changes in the microbiota (e.g., Dubosiella) can impact the liver metabolites (e.g., TGs) through alterations in fecal metabolites (e.g., LPCs). These findings provide a theoretical reference for the long-term effect of arsenic exposure on liver lipid metabolism.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

Abstract 5811: Ovarian cancer cell fate is regulated by the balance between saturated and unsaturated fatty acids

Abstract Background: Fat represents an important source of energy for ovarian cancer (OC) cells and is supplied either through import from the tumor milieu or via de novo lipogenesis. During fast tumor growth, when nutrients are scarce, lipogenesis becomes the primary source of fatty acids. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in this pathway converts saturated (SFAs) into unsaturated fatty acids (UFAs) and is highly expressed in OC. The goal of this study was to determine how the balance between SFAs and UFAs tightly controlled by SCD, regulates OC cell survival and tumorigenicity. Methods: SCD was knocked down by shRNA or inhibited by using the small molecule CAY10566 and global effects on the lipidome and transcriptome were examined by targeted and untargeted lipidomics, stimulated Raman spectroscopy (SRS), and RNA sequencing. In OC cells in which SCD was blocked or knocked down, the effects of exogenous SFAs and UFAs on cell survival and endoplasmic reticulum (ER) stress pathway were assessed by using annexin V staining, XBP1 splicing assay and Western blotting of PERK/eIF2α/ATF4. Tumorigenicity was assessed by using an intraperitoneal (i.p.) xenograft model in nude mice. Results: RNA-seq analysis of SCD knockdown cells cultured under low serum conditions revealed activation of ER stress response pathways. Targeted lipidomics and SRS microscopy showed downregulation of UFAs vs. SFAs, while untargeted lipidomics discovered decreased fatty acyl chain plasticity among phosphatidylcholines. Activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes was observed in cells accumulating SFAs. Stiff and disorganized ER membrane was detected by electron microscopy and Raman spectroscopy. Annexin V staining demonstrated apoptosis of OC cells under long-term mild ER stress or short-time severe ER stress induced by increased levels of SFAs. ER stress and apoptosis were rescued by addition of UFAs. In vivo SCD knockdown suppressed tumor growth and treatment with the SCD inhibitor CAY10566 reduced the number of metastases and the volume of ascites in mice fed with SFA enriched diet, but not in mice fed a balanced diet. Conclusion: Our data support that OC cells are highly susceptible to unbalanced intracellular SFAs/UFAs, undergoing ER stress and apoptosis in the presence of excessive intracellular SFAs. SCD inhibition coupled with a diet rich in saturated fats decreased cancer progression in vivo. These results support SCD as a key regulator of cancer cell fate during metabolic stress in growing tumors and point to new treatment strategies targeting lipid metabolism. Citation Format: Guangyuan Zhao, Yuying Tan, Horacio Cardenas, David Vayngart, Yinu Wang, Christina Ferreira, Ji-Xin Cheng, Daniela Matei. Ovarian cancer cell fate is regulated by the balance between saturated and unsaturated fatty acids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5811.

Structural and physiological effects of chromium uptake in the seagrass Halophila stipulacea

This work aims to provide insight on the effects of chromium (Cr) uptake in seagrasses at environmentally relevant exposure concentrations; information on this issue can further increase the utility of marine angiosperms as indicators of trace metal contamination. Cr uptake kinetics, effects on physiological and structural cell traits, and the uptake-effects relationship in Halophila stipulacea leaves incubated in 0.01–10 mg L−1 (0.2–200 μM) of Cr (VI) were examined. An initial uptake was followed by a decrease at 0.01–0.5 mg L−1 (0.2–10 μM); at higher exposures, the fit of uptake data to a Michaelis-Menten-type equation was significant (p < 0.001). The mean tissue concentration over the exposure period, and the highest tissue concentration attained both increased as exposure concentration (Cw) increased from 0.05 to 10 mg L−1 (1 to 200 μM); uptake rate generally increased with Cw. Cytotoxic effects occurred even at 0.01 mg L−1 (0.2 μM). Actin filaments (AFs) and endoplasmic reticulum (ER) were impacted in a dose- and time- dependent pattern, while microtubule integrity was not markedly affected even at the highest exposure. AF disruption in differentiating cells initiated at 0.01 mg L−1 (0.2 μM), while ER disorganization did so at 0.1 mg L−1 (2 μM); regression equations described the relationships of Cw vs. time required for effect onset, and of Cw vs. AF disturbance progress. Elevated levels of hydrogen peroxide (H2O2) were detected on the 2d. In addition, a significant reduction in epidermal cell viability appeared at 5–10 or 10 mg L−1 (100–200 or 200 μM), depending on the leaf age. The lowest tissue concentration associated with effect onset was 31.05 μg g−1 dry wt (2d, 5 mg L−1); at lower exposures, higher tissue concentrations did not coincide with impact, implying that the uptake rate rather than the tissue concentration relates to the effect onset. The above suggest that (a) Cr (VI) may pose a high risk to seagrass meadows, (b) H. stipulacea can act as a bioindicator of ambient Cr (VI) bioavailability, mainly at elevated exposure concentrations, (c) AF and ER disturbances in seagrass leaf cells could be considered as sensitive biomarkers of Cr (VI)-induced stress, (d) regression equations describing the relationships Cw vs. time required for effect onset and Cw vs. effect progress increase the utility of AF and ER impairment as biomarkers, and (e) the time frame of the exposure should be considered in the interpretation of tissue residues. Our findings improve the utility of seagrasses as indicators of trace metal contamination and could be utilized in the overall effort for seagrass meadows conservation.

Long-term uninterrupted enzyme replacement therapy prevents liver disease in murine model of severe homocystinuria.

Classical homocystinuria (HCU) is an inborn error of metabolism caused by loss of cystathionine β-synthase (CBS) activity with the concomitant buildup of homocysteine. In knockout (KO) mice, a mouse model of HCU, complete lack of CBS is neonatally lethal. Administration of OT-58, an enzyme therapy for HCU, during the first 5 weeks of life rescued KO mice survival by preventing liver disease. Here, we studied the impact of a long-term uninterrupted OT-58 treatment or its absence beyond the neonatal period on liver pathology and metabolism. Plasma and liver metabolites of KO mice on OT-58 treatment were substantially improved or normalized compared with those receiving vehicle. Increased plasma activities of alanine aminotransferase and aspartate aminotransferase of vehicle-injected KO mice suggested the progression of liver damage with age and lack of treatment. At 3 months of age, liver histology showed no signs of hepatopathy in both vehicle- and OT-58-treated KO mice. However, moderate to severe liver disease, characterized by steatosis, hepatocellular necroses, disorganized endoplasmic reticulum, and swollen mitochondria, developed in 6-month-old vehicle-injected KO mice. KO mice on OT-58 treatment remained asymptomatic and were indistinguishable from age-matched healthy controls. Long-term uninterrupted OT-58 treatment was essential to prevent severe liver disease in the KO mouse model of HCU.

GATA6 Controls Insulin Biosynthesis and Secretion in Adult β-Cells.

GATA4 and GATA6 play essential, but redundant, roles in pancreas formation in mice, and GATA6 mutations cause pancreatic agenesis in humans. GATA6 mutations have also recently been linked to adult-onset diabetes, with subclinical or no exocrine insufficiency, suggesting an important role for GATA6 in human β-cell physiology. To investigate the role of GATA6 in the adult endocrine pancreas, we generated mice in which Gata6 is specifically inactivated in the pancreas. These mice develop glucose intolerance. Islets deficient in GATA6 activity display decreased insulin content and impaired insulin secretion. Gata6-deficient β-cells exhibit ultrastructural abnormalities, including increased immature insulin granules, swollen mitochondria, and disorganized endoplasmic reticulum. We also demonstrate that Pdx1 expression in adult β-cells depends on GATA sites in transgenic reporter mice and that loss of GATA6 greatly affects β-cell-specific gene expression. These findings demonstrate the essential role of GATA6 in β-cell function.

Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos.

The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3-dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo.

NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization

Nuclear transcription factor-Y (NF-Y), a key regulator of cell-cycle progression, often loses its activity during differentiation into nonproliferative cells. In contrast, NF-Y is still active in mature, differentiated neurons, although its neuronal significance remains obscure. Here we show that conditional deletion of the subunit NF-YA in postmitotic mouse neurons induces progressive neurodegeneration with distinctive ubiquitin/p62 pathology; these proteins are not incorporated into filamentous inclusion but co-accumulated with insoluble membrane proteins broadly on endoplasmic reticulum (ER). The degeneration also accompanies drastic ER disorganization, that is, an aberrant increase in ribosome-free ER in the perinuclear region, without inducing ER stress response. We further perform chromatin immunoprecipitation and identify several NF-Y physiological targets including Grp94 potentially involved in ER disorganization. We propose that NF-Y is involved in a unique regulation mechanism of ER organization in mature neurons and its disruption causes previously undescribed novel neuropathology accompanying abnormal ubiquitin/p62 accumulation.

Inactivation of CBF/NF-Y in postnatal liver causes hepatocellular degeneration, lipid deposition and endoplasmic reticulum stress

We previously demonstrated that CBF activity is needed for cell proliferation and early embryonic development. To examine the in vivo function of CBF in differentiated hepatocytes, we conditionally deleted CBF-B in hepatocytes after birth. Deletion of CBF-B resulted in progressive liver injury and severe hepatocellular degeneration 4 weeks after birth. Electron microscopic examination demonstrated pleiotropic changes of hepatocytes including enlarged cell and nuclear size, intracellular lipid deposition, disorganized endoplasmic reticulum, and mitochondrial abnormalities. Gene expression analyses showed that deletion of CBF-B activated expression of specific endoplasmic reticulum (ER) stress-regulated genes. Inactivation of CBF-B also inhibited expression of C/EBP alpha, an important transcription factor controlling various metabolic processes in adult hepatocytes. Altogether, our study reveals for the first time that CBF is a key transcription factor controlling ER function and metabolic processes in mature hepatocytes.

Characterization of the Withdrawal Phase in a Porcine Donation after the Cardiac Death Model

Transplantation of donation after cardiac death (DCD) livers has higher rates of organ failure and complications, specifically ischemic biliary injuries. Reported large animal DCD models all employ active means to halt circulation, contrary to human DCD protocol. We report a DCD porcine model in which the animal passively progresses to cardiac death, thereby more closely mimicking human DCD scenario. Sixteen Yorkshire pigs (10 females, 6 males, 30-45 kg) had a mean time of 26:19 min ± 14:14 from withdrawal of ventilatory support (WVS) to circulatory arrest and 44:38 min ± 16:37 from WVS to electrical standstill. Cessation of hepatic flow (HF) occurred well before electrical standstill (22:15 min ± 10:09), previously not described in human or animal DCD. Histologically comparing livers from our DCD model demonstrated a dramatic increase in hepatocyte vacuolization, disorganization of endoplasmic reticulum, formation of mitochondrial inclusions and apoptosis compared with control specimens. Subtle changes were also evident in biliary epithelial cells (BEC). This results in severe cellular changes before reperfusion. Early histologic evidence suggests that there is severe hepatocyte and biliary cell disruption in our DCD model. Further research using this model may provide a deeper understanding of the pathophysiology of the DCD liver.

Characterization of the Properties of a Novel Mutation in VAPB in Familial Amyotrophic Lateral Sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.

Ultrastructural changes in rat livers perfused in vitro and in vivo with a high dose of methotrexate.

Methotrexate is an antifolate that is widely used in the treatment of malignant tumours and other diseases. The present study was undertaken to examine the short-term effects of high doses of methotrexate (HD-MTX) on the ultrastructure and metabolic activity of isolated rat livers. The authenticity of the drug-induced changes was substantiated by the concomitant use of in vivo experiments. Isolated rat livers were infused with HD-MTX via the portal vein for 3 hours (total dose for each liver 2000 mg). For in vivo experiments, each rat received a single intravenous injection of a maximum tolerated dose of MTX (100 mg/kg body weight) that allowed the animals to survive for 3 days. At the end of each experimental period, MTX-treated and control livers were processed for light microscopy (LM), scanning (SEM) and transmission electron (TEM) microscopy. Oxygen consumption and thyroxine metabolism were measured in treated and control isolated livers. With the exception of a few minor differences, the structural changes in the hepatocytes after MTX treatment in vitro and vivo were similar. There were focal changes consisting of disruption of normal hepatic plates and swelling and vacuolation of the hepatocytes, with no clear evidence of restriction to a specific hepatic zone. SEM revealed striking changes in the plasma membrane, the microvillar system, intercellular junctions and the sinusoidal endothelium. TEM revealed disorganized endoplasmic reticulum, dispersion of the polyribosomes, a variety of mitochondrial changes, and glycogen redistribution. In MTX-treated isolated rat livers, the uptake of tetraiodothyronine (T4) was not affected, but triiodothyronine (T3) release was impaired. Oxygen consumption was increased in livers treated with MTX. Employing an organotypic liver perfusion model in conjunction with the in vivo experiment and the use of SEM, TEM and hepatic thyroxine measurements, this investigation revealed that infusion of HD-MTX induced early ultrastructual changes in cell membrane, intercellular junctions and cell organelles and disturbance in the functional integrity of the hepatocytes in isolated rat liver.

Effect of endothelin-1 receptor antagonists on histological and ultrastructural changes in the pancreas and trypsinogen activation in the early course of caerulein-induced acute pancreatitis in rats

To assess the effect of non-selective ET(A/B) (LU 302872) and selective ET(A) (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP. Male Wistar rats with caerulein-induced AP, lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w. of each antagonist. Edema, inflammatory infiltration, necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase, and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates. In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P<0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82+/-0.06 at higher dose (P<0.05) vs 0.58+/-0.06 in untreated AP. The non-selective antagonist increased slightly the vacuolization score to 2.41+/-0.07 at higher dose (P<0.01) vs 1.88+/-0.08 in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum, autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups. %FAT/TPT in untreated AP increased about four times (18.4+/-3.8 vs 4.8+/-1.3 in control group without AP, P<0.001). Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation. The treatment with endothelin-1 receptors (non-selective ET(A/B) and selective ET(A)) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.

Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: protective role of 70 kDa heat shock protein.

We explored the reproductive toxicity of argemone oil and its principal alkaloid fraction in transgenic Drosophila melanogaster (hsp70-lacZ) Bg(9). The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Freshly eclosed first instar larvae of transgenic Drosophila melanogaster were transferred to different concentrations of argemone oil and its alkaloid fraction contaminated food. Virgin flies that eclosed from the contaminated food were pair-mated to look into the effect on reproduction. The study was further extended by investigating hsp70 expression and tissue damage in larval gonads, genital discs, and reproductive organs of adult fly. Our results showed that argemone oil was more cytotoxic than its principal alkaloid fraction. Moreover, it was the male fly that was more affected compared to its opposite number. The accessory glands of male reproductive system of the fly, which did not express hsp70, exhibited severe damage as evidenced by Trypan blue staining. This prompted us to explore the ultrastructural morphology of the gland, which showed acute signs of necrosis in both the cell types as evident by necrotic nuclei, higher vacuolization, and disorganized endoplasmic reticulum, decrease in the number of Golgi vesicles and disorganized, loosely packed filamentous structures in the lumen of the accessory gland, at the higher concentrations of the adulterant. The study showed the reproductive toxicity of argemone oil and its alkaloid fraction in transgenic Drosophila melanogaster and further confirmed the cytoprotective role of hsp70.

Carbon tetrachloride-induced morphologic alterations in isolated rat hepatocytes

Isolated rat hepatocytes were exposed to CCl 4 in doses commonly used in in vitro studies and for which we have provided biochemical evidence would induce solvent injury. Rapidly evolving morphologic alterations were observed in the plasma membrane, endoplasmic reticulum, and mitochondria. Swelling and fusion of surface microvilli with formation of blebs were particularly prominent and occurred within 2 min of exposure. Blebs regressed in some hepatocytes without evidence of cell death, when these cells were exposed to CCl 4 under conditions promoting its evaporation. Disorganization of endoplasmic reticulum and mitochondrial injury were also prominent early findings. Rapid appearance of diffuse ultrastructural alterations in isolated hepatocytes exposed to CCl 4 is consistent with nonspecific membrane injury induced by solvent effects.

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice.

Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued using in vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study membrane-bound polysomes isolated from the livers of newborn albinos were 55% (c3H/c3H strain) and 62% (c14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)+-RNA isolated from albino livers assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.

The ultrastructure and function of the cells in lymph following antigenic stimulation.

When a lymph node receives an antigenic stimulus the cell population in the efferent lymph changes and large basophilic cells appear. During a secondary immune response cells of this type may account for over 50% of the cells present in lymph. When examined by electron microscopy, many of these cells were found to be primitive undifferentiated blast cells with many free ribosomes in their cytoplasm and only an occasional piece of endoplasmic reticulum. Their nuclear chromatin was sparse and the nuclei contained several nucleoli. Many other cells which were judged to be more differentiated had large numbers of ribosomes arranged in clusters which took the form of rosettes or spirals. These cells also had more ergastoplasm but this occurred usually in the form of short pieces of disorganized endoplasmic reticulum. No cells with the ultrastructure of classical plasma cells were found in efferent lymph although these cells were abundant in the stimulated lymph nodes. It was shown that when the lymph which contained these cells was collected quantitatively no systemic immunity developed even though a vigorous immune response took place in the lymph node with the formation of many plasma cells. Failure of the systemic immune response to develop could not be explained merely in terms of the loss of antibody. It was concluded that these basophilic cells rather than antigen are responsible for propagating the immune response throughout the body and that they depend on an intact lymphatic pathway for their immediate transport. This view was supported by experiments which showed that these cells are capable of initiating immune responses in other lymph nodes of the same animal and of transferring active immunity between chimeric twins. The most likely explanation of these results is that the basophilic lymphoid cells carry out their messenger function by developing into plasma cells at sites remote from the site at which antigen is localized. However this has yet to be proven and the possibility remains that these mobile, highly motile, RNA-rich cells may express their messenger function by transferring information to other effector cells.