Ultrastructure of a Mobile Threadlike Tissue Floating in a Lymph Vessel

Observations of the primo vascular system (PVS) floating in lymph ducts were reported by various groups. There have been, however, no studies on the ultrastructure of the entire cross section of a primo vessel (PV) inside a lymph vessel with a transmission electron microscope (TEM). In the current study we took the TEM images of a cross section of the PV inside a lymph vessel. We used the Alcian blue staining method for the finding of the target PV in a lymphatic vessel by injecting the dye into the inguinal lymph nodes. The stained PV was harvested together with the lymph vessel and some parts of the specimens were used for studying with optical microscopes. Some other parts were treated according to a standard protocol for TEM. As the results the TEM study revealed the loosely distributed collagen fibers with plenty of empty spaces and the lumens with the endothelial nuclei. It turned out to be very similar to the ultrastructure of the PVs observed on the surfaces of internal organs. It also showed how compactly the PV is surrounded with lymphocytes. In conclusion, the detailed morphological features like the distribution of fibers in the PV were revealed and shown to be similar to another kind of the PV on the surfaces of internal organs

Regulator of G protein signaling 1 suppresses CXCL12-mediated migration and AKT activation in RPMI 8226 human plasmacytoma cells and plasmablasts.

Migration of plasma cells to the bone marrow is critical factor to humoral immunity and controlled by chemokines. Regulator of G protein signaling 1 (RGS1) is a GTPase-activating protein that controls various crucial functions such as migration. Here, we show that RGS1 controls the chemotactic migration of RPMI 8226 human plasmacytoma cells and human plasmablasts. LPS strongly increased RGS1 expression and retarded the migration of RPMI 8226 cells by suppressing CXCL12-mediated AKT activation. RGS1 knockdown by siRNA abolished the retardation of migration and AKT suppression by LPS. RGS1-dependent regulation of migration via AKT is also observed in cultured plasmablasts. We propose novel functions of RGS1 that suppress AKT activation and the migration of RPMI 8226 cells and plasmablasts in CXCL12-mediated chemotaxis

Observation of a Flowing Duct in the Abdominal Wall by Using Nanoparticles.

The primo vascular system (PVS) is being established as a circulatory system that corresponds to acupuncture meridians. There have been two critical questions in making the PVS accepted as a novel liquid flowing system. The first one was directly to show the flow of liquid in PVS and the second one was to explain why it was not observed in the conventional histological study of animal tissues. Flow in the PVS in the abdominal cavity was previously verified by injecting Alcian blue into a primo node. However, the tracing of the dye to other subsystems of the PVS has not been done. In the current work we injected fluorescent nanoparticles (FNPs) into a primo node and traced them along a primo vessel which was inside a fat tissue in the abdominal wall. Linea alba is a white middle line in the abdominal skin of a mammal and a band of fat tissue is located in parallel to the linea alba in the parietal side of the abdominal wall of a rat. In this fat band a primo vessel runs parallel to the prominent blood vessels in the fat band and is located just inside the parietal peritoneum. About the second question on the reason why the PVS was not in conventional histological study the current work provided the answer. Histological analysis with hematoxyline and eosine, Masson's trichrome, and Toluidine blue could not discriminate the primo vessel even when we knew the location of the PVS by the trace of the FNPs. This clearly explains why the PVS is hard to observe in conventional histology: it is not a matter of resolution but the contrast. The PVS has very similar structure to the connective tissues that surround the PVS. In the current work we propose a method to find the PVS: Observation of mast cell distribution with toluidine blue staining and the PN has a high density of mast cells, while the lymph node has low density

LPS increases RGS1 expression in RPMI 8226 cells.

<p>A) Quantitative PCR analysis of RGS1 expression in TLR ligand-treated RPMI 8226 cells. Cultured cells were stimulated with PGN (peptidoglycan) (10 μg/mL; TLR2), poly (I:C) (1 μg/mL; TLR3), LPS (10 ng/mL; TLR4), flagellin (50 ng/mL; TLR5), R848 (3 μM; TLR7), and CpG-B (2 μM; TLR9). After stimulation, cells were harvested and quantitative PCR analysis was performed, as described in the Materials and Methods section. Data show the relative expression levels of RGS1 normalized to the relative S18 expression level of each mRNA (*<i>p</i> <0.05). B) RGS1 expression in LPS-treated RPMI 8226 cells. Cells were cultured in the presence or absence of LPS for 18 hours. To examine the specificity of the RGS1 antibody, the peptide competition assay was performed by addition of RGS1 peptide (1:1 molar ratio with antibody) in antibody binding reaction of ICC. Images shown are the representatives taken under 1000× objective magnification by a confocal microscope.</p

LPS reduces CXCL12-mediated migration via RGS1-AKT in plasmablasts.

<p>GC-B cells were cultured in combination with IL-2, IL-4, and IL-21 for 7 days and differentiated into plasmablasts. The cultured plasmablasts were stimulated with 10 ng/mL LPS for 18 hours. RGS1 expression and AKT phosphorylation were assessed in LPS-treated plasmablasts, in addition to assessment using the transwell migration assay. A) CXCR4, CD38, CD19, and Ki-67 expression levels in the cultured plasmablasts. B) Expressions of Bcl-6 and Blimp-1 were measured by real-time PCR at GC-B cells and cultured plasmablast. C) Amount of secreted immunoglobulin was measured at culture day4 and culture day 7. D) After 7 days of culture, purified GC-B cells differentiated to plasmablasts. The cytoplasm is abundant and the nucleus characteristically shows the eccentric position. The nucleus shows fine reticular or mildly blocked chromatin pattern. These plasmablasts show strong cytoplasmic staining for the kappa or the lambda light chain immunocytochemical staining. (H&E, Hematoxylin and eosin stain × 1,000; Kappa, anti-kappa light chain, × 1,000; Lamda, anti-lambda light chain, × 1,000). E) Plasmablasts demonstrate high migratory properties towards CXCL12. F) LPS treatment induces RGS1. G) LPS reduces CXCL12-mediated transwell migration. H) AKT-specific inhibitors reduce migration. I) LPS reduces CXCL12-mediated AKT phosphorylation. Representative western blot of four repetitive experiments is shown. Quantification of phospho-AKT expression was shown in Graph. Phospho-AKT expression was normalized to control levels and corrected for loading differences using GAPDH. J) Intracellular staining of phospho-AKT showed the reduction of the phospho-AKT by LPS treatment. Representative data of two repetitive experiments was shown. Average mean fluorescence intensity (MFI) was shown in graph (*<i>p</i> <0.05; **<i>p</i> <0.005***<i>p</i> <0.0005).</p

LPS reduces CXCL12-mediated migration by RGS1 in RPMI 8226 cells.

<p>RPMI 8226 cells (with or without LPS treatment) were assessed using the transwell migration assay. The upper chamber contained 1 × 10⁵ cells in 100 μL media, and the lower chamber contained 600 μL media. A) LPS reduced CXCL12-mediated transwell migration. Cells were cultured in the presence or absence of LPS for 18 hours before assessment using the migration assay. Cells were allowed to migrate for 5 hours in the absence or presence of 100 ng CXCL12 in the lower chamber of the transwell assay. After 5 hours of migration, 300 μL of the lower chamber media were removed, and cells were counted using FACS and PI. B) CXCR4 expression in LPS-treated RPMI 8226 cells. After 18 hours of incubation with LPS, 2 × 10⁵ cells were harvested, resuspended, and incubated with CXCR4-APC or mouse IgG-APC for 20 minutes on ice. The surface CXCR4 expression was then analyzed using FACS. C) In total, 2 × 10⁶ cells were transfected using a Neon electroporator with siRNA-targeting RGS1 (RGS1i) or nontargeting scrambled RNA (Scr). Three days after electroporation, cells were stimulated with LPS for 18 hours. The reduction in RGS1 RNA expression was assessed using quantitative PCR and RGS1 protein expression was accessed with western blot by anti-RGS1 antibody. D) Transwell migration of Scr-transfected cells and RGS1i-transfected cells, with or without LPS stimulation (*<i>p</i> < 0.05; ***<i>p</i> < 0.0005; N/S: nonsignificant).</p

Histological analysis of the PV, peritoneum and fascia.

<p><b>A:</b> The position of the tissue block for the cross section. <b>B:</b> The spot of fluorescence (arrow) due to FNPs is the position of the PV. Its size is 10 μm and 150 μm away to the left from the large blood vessel. <b>C:</b> The H&E staining barely revealed the spot of the PV (arrow) just inside the parietal peritoneum. This figure showed that the PV and the surrounding connective tissue of the parietal peritoneum are not distinguishable with H&E. The deep fascia and the parietal peritoneum are barely distinguishable. Muscle (M) is clearly distinguished by color. B &C are the same sections. <b>D</b>: Another section showed the fluorescence spot of the PV (arrow). <b>E</b>: The Mason’s trichrome staining cannot distinguish the PV (arrow) and the parietal peritoneum. It can clearly distinguish the parietal peritoneum and the deep fascia. Muscle (M) is also well distinguished. D &E are the same sections.</p

Phase contrast microscope images of the PVS.

<p>A: The fluorescent image of the FNPs that were injected at a PN located about the CV4 and flowed in a PVS buried in the adipose tissue of the AWFB. It flowed up to the CV 14 and reemerged to the abdominal cavity toward the liver surface. The flow line was barely visible under the stereo fluorescence microscope. B & C: Phase contrast microscope images of the bright mode (B) and fluorescent mode (C) of the boxed region in (A). The PV (dashed arrow) running parallel to and above the blood vessel (two arrows) is hardly visible in (B) but clearly observable with fluorescence of FNPs in the panel (C). This primo vessel is the first observation of the so called extra vascular PVS. It runs closely along the blood vessel. The AWFB is clearly seen in (B) and its boundary is depicted with two curves in (C). The boundary of the abdominal wall fat band is indicated with two yellow curves. 40x. D & E: The PN is not noticeable without the fluorescence in (D), but manifestly appears with fluorescent view in the panel (E). The size of the PN was 250 μm. The fluorescent nanoparticles were highly concentrated in the PN. 40x</p

The anatomical position of the novel flowing duct in the abdominal wall fat band.

<p>A: Schematic illustration showing the location of linea alba and the conception vessel CV in the abdominal skin side. The broken line is the surgery cutting line of the abdominal wall. The line is in the right hand side from the linea alba in order to avoid cutting the PVS in the AWFB. The FNPs that were injected to a PN entered the AWFB and appeared at the terminal point to be continued to the PVS on the liver surface. B: The blood vessels in the AWFB inside the parietal peritoneum of the abdominal wall. The locations CV 8 to 14 are mere markings for positional references and not real CV-acupoints. Note that the CV8 corresponds to the umbilicus and the parietal peritoneum continues down to the ligament wrapping the bladder. C: A PV (arrow) emerged from the AWFB (double arrows) was raised tautly with a forceps.</p