The involvement of omentum and its milky spots in the dynamics of peritoneal macrophages

The investigation has been carried out on stimulated and unstimulated peritoneal cavities of rats. China ink and Corynebacterium parvum were injected i.p. both as peritoneal stimuli and markers. Omenta were picked up at time intervals beginning with 10 min and up to seven days after the i.p. injection. The light and electronmicroscopic investigation showed after 10-30 minutes labeled macrophages stuck as monolayers on some peritoneal areas corresponding to the milky spots which developed in size and number. Days after the i.p. injection the labeled macrophages were found deeper in the milky spots. After the fourth day they appeared in the regional lymph nodes. The milky spots contained also large lymphocytes and plasma cells. The results suggest that milky spots are not only places of resident macrophages development and release in the peritoneal cavity but also their exit pathways. Therefore the omentum leads the traffic of peritoneal macrophages. The developed milky spots play also the role of lymphoid structures providing grounds for macrophage-lymphocyte contacts.     

Milky spots in the greater omentum are predominant sites of local tumour cell proliferation and accumulation in the peritoneal cavity

The role that milky spots in the greater omentum play in tumour cell spread in the peritoneal cavity is presently not fully understood. To study whether intraperitoneally injected tumour cells appear preferentially in milky spots of the greater omentum and to study the changes in the greater omentum, and especially in the cell population of milky spots after tumour cell infiltration, the following study was performed. A detailed temporal sequences of changes in morphology and cellular composition in milky spots of the greater omentum of Wag/Rij rats 5, 15, 30, 60 min, 2, 4, 8, 16, 24 h, 2, 4, 8 days and 2 and 4 weeks after intraperitoneal administration of 2.0 × 10⁶ CC 531 tumour cells was investigated by light microscopy and electron microscopy (pre-embedding labelling). Our data showed that the milky spots in the greater omentum were the sites to which tumour cells migrated preferentially from the peritoneal cavity. The tumour cells infiltrated the milky spots and formed clusters within. The cellular population in milky spots reacted by a very rapid influx of young macrophages during the first hour and an increase of the total number of cells (P < 0.01). After 4 h tumour cells were also located on the greater omentum outside the area of the milky spots. Around these tumour cell deposits, new milky spots are formed, which increased the total number of milky spots. The cells present in milky spots are not capable of reversing the growth of tumours and finally a solid omental cake of tumour cells is formed. Received: 30 June 1998 / Accepted: 3 September 1998     

Biodefense function of omental milky spots through cell adhesion molecules and leukocyte proliferation

To evaluate the immunological functions of the greater omentum in the peritoneal cavity, the localization of cell adhesion molecules (CAMs) on mesothelial cells and leukocytes in the omental milky spots were studied in normal and lipopolysaccharide (LPS)-stimulated mice by means of immunoelectron microscopy. The milky spots featured numerous leukocytes among the dome-shaped mesothelial cells, even in the normal stable state. Leukocyte integrins LFA-1, Mac-1, and VLA-4 were preferentially localized to microvilli and ruffles of macrophages and lymphocytes. The mesothelial cells of the milky spots showed higher ICAM-1 levels than did those of other omental regions, and fibronectin was detected in the stomata. The number of leukocytes markedly increased following an increase in proliferating cell nuclear antigen (PCNA)-positive cells in the milky spots after LPS stimulation. The mesothelial cells contained VCAM-1 newly restricted to the microvilli and increasing amounts of ICAM-1. These results show that the omental milky spots are active sites for leukocyte migration and peritoneal leukocyte supply because of the presence of adhesion molecules and active cell proliferation. Proliferative active leukocytes and those that have migrated from vessels pass through the stomata via an interaction of VLA-4 and fibronectin, adhere to the microvilli of the activated mesothelial cell surface as the result of an interaction between ICAM-1/VCAM-1 and integrins, and exude into the peritoneal cavity. Much of the exudation and adhesion of leukocytes seen in the milky spots of LPS-stimulated mice may be attributable to an increase in cell proliferation and in the amounts of ICAM-1 and VCAM-1.     

Cellular subsets of the milky spots in the human greater omentum

Summary The cellular composition of the human milky spots was investigated on surgically removed specimens of the greater omentum of three 8-month-old infants operated on for neuroblastoma. Monoclonal antibodies and immunohistochemical methods for recognition of macrophages, B-lymphocytes and T-lymphocytes and toluidine-blue staining for mast cells were used. The mean number of cells in one milky spot amounted to 570±33. This cell population was composed of 47.5% macrophages, 29.1% B-lymphocytes, 11.7% T-lymphocytes and 6.1% mast cells. Since inflammation was absent in the material investigated, the numerical data found in the present paper could be regarded as representative cell levels of normal milky spots.     

Milky spots in the human greater omentum. Macroscopic and histological identification

Macroscopic and histological investigations were made from surgical specimens demonstrating milky spots in the human greater omentum from subjects of various ages. The milky spots in the human greater omentum appear as tiny, cotton-wool-like masses to the naked eye. Histologically, the milky spots consisted mainly of many macrophages with diffuse cytoplasmic esterase reaction products and esterase-negative B lymphocytes surrounding the vascular networks. Macrophages phagocytosed many carbon particles which were introduced as a carbon suspension during the operation. The vascular networks were blood capillary convolutions with a glomerular shape. Silver impregnation showed the delicate networks of reticular fibers which constitute the framework of the organ. The number of milky spots was highest in infancy and gradually decreased with age.     

The milky spots on the chest wall in newborns

The histologic properties of pleural adipose organs were studied in 14 newborns. These organs contain milky spots, in which lymphocytes, macrophages and plurivacuolated fat cells are present. The milky spots have a mesothelial covering, persist to the age of 9 months and seem to act as defence devices and a site of fluid exchange.     

A rapid and simple method for visualizing milky spots in large fixed tissue samples of the human greater omentum

ABSTRACT

Milky spots are unique lymphoid structures in the greater omentum that participate in both immune homeostasis of the peritoneal cavity and formation of omental metastases. We developed a rapid and simple staining method to enable macro- or stereomicroscopic identification of these miniscule structures in large samples of fixed human greater omentum. By immersing approximately 6 × 4 cm samples of omental tissue in hematoxylin, these samples could be evaluated quickly for the presence of milky spots. We used an alum hematoxylin variant containing 1 g hematoxylin, 50 g aluminium ammonium sulfate, 0.2 g sodium iodide, 1 g citric acid and 50 g chloral hydrate. This staining method enabled us to determine the number, location, dimensions and topographical relation of milky spots to other structures. Our method also facilitates isolation of milky spots for further investigation. Hematoxylin imparts a blue color to the milky spots, which remains in place during further processing for paraffin embedding. This enabled easy recognition of milky spots during transfer through various solutions and permitted selection of relevant paraffin slides prior to additional staining.     

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.     

Insufficient ability of omental milky spots to prevent peritoneal tumor outgrowth supports omentectomy in minimal residual disease

Background: The greater omentum is frequently involved in the course of gastrointestinal and ovarian tumors. Therefore, common practice in surgical treatment for especially gastric and ovarian cancer includes removal of the greater omentum. Paradoxically, many immune cells, such as macrophages that accumulate in so-called milky spots, reside within the omentum and are cytotoxic against tumor cells ex vivo. Consequently, omental macrophages might play an important role in killing tumor cells, and may hereby prevent development into local peritoneal recurrences. In the present study, we therefore evaluated the role of the omentum and the clinical relevance of omentectomy in minimal residual disease (MRD). Methods: Tumor cell dissemination patterns on the omentum in a rat model were examined using DiI-labelled CC531s tumor cells. Additionally, intra peritoneal (i.p.) tumor load was investigated in rats that underwent omentectomy or sham laparotomy followed by i.p. injection of CC531s cells on day 21, which represented MRD. Results: At 4 h post injection, tumor cells predominantly adhered on milky spots. Number of cells thereafter declined rapidly suggesting initial tumor killing functions in these specific immune aggregates. Despite initial reduction observed in milky spots, numbers of tumor cells however increased at fatty tissue stripes that border the omentum. This indicated proliferation at these locations, which corresponded to macroscopic observations of the omenta on day 21 after tumor cell injection. Omentectomy resulted in reduced intra-abdominal tumor load, which was completely attributable to the absence of the omentum, as tumor development did not differ on other sites. Even in the MRD group microscopic clusters of tumor cells located in the omentum eventually developed into macroscopic nodules.Conclusion: Since the ability of omental milky spots is, even in MRD, insufficient to prevent intra abdominal tumor outgrowth, omentectomy, which reduces tumor load, is recommended in surgical treatment of intra abdominal tumors that are prone to disseminate intraperitoneally.     

Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection

Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.     

Blood supply of the greater omentum in sheep and goats

The course and shape of the arteries and veins which - as side branches of the A. and V. gastroepiploica and the A. and V. epiploica - supply the greater omentum of sheep and goat were demonstrated by means of the india ink method and by stained membrane preparations. The terminal vascular bed shows the principle of the s.c. bridgeformation, consisting of a preferential channel and parallel connected capillaries, which are encircled with precapillary sphincters. The capillaries comprise a continuous endothelium. There are capillary convolutes - sometimes furnished with a fenestrated endothelium - which are parts of the milky spots. Other capillary convolutes not belonging to the milky spots contain a nonfenestrated endothelium.     

The development of lymphatic follicles in the omentum after intraperitoneal stimulation of rats

China ink and typhoid vaccine were used as peritoneal stimuli injected in single doses. The subsequent omental reaction was studied after several time intervals: 6 and 12 hours, 1, 2, 3, 4 and 8 days, 3 and 6 weeks. The omental reaction featured as lymphatic nodules connected to milky spots was compared with a primary immune response. The ultrastructure and development of the characteristic cells (lymphocytes, lymphoblasts, dendritic reticulum cells, tingible body macrophages and plasma cells) was investigated throughout the period from 6 h to 6 weeks. A special attention was devoted to ultrastructural and functional features of plasma cells.     

Mesothelial stomata overlying omental milky spots: Scanning electron microscopic study

Summary The presence in the rat omentum of intercellular pores (the classical stomata of von Recklinghausen) between the mesothelial cells overlying aggregates of lymphoreticular cells (the classical milky spots of Ranvier) and the apparent migration of lymphocytes through these stomata were recorded for the first time by scanning electron microscopy. Previous studies on passage of cells across the peritoneum and omentum used experimentally administered cells, while in the present study no cells were administered to the rats and their own lymphocytes were observed in situ. The possible role of lymphocytes in the peritoneal cavity is also briefly discussed.     

Quantitative histochemical studies of the effect of levamisole on splenic T-cells in thymectomized Bufo bufo larvae

Summary The ability of levamisole (LVS) to correct the effect of thymectomy on splenic T-cell population in Bufo bufo larvae was tested. Using the nonspecific acid -naphthyl acetate esterase (ANAE) technique, the T-lymphocytes displayed a characteristic reddish brown spot (T-pattern). The total number of white cells per spleen and the number of T-pattern cells per 100 splenic white cells were determined. In addition, differential counts of the T-patterns were made on the basis of the size of their spots.Thymectomized larvae in comparison to sham-operated larvae posessed a lower total number of splenic white cells with a similar proportion of T-pattern cells, but a lower proportion of cells with a large T-pattern. After seven days of LVS treatment, the thymectomized larvae showed an increased total number of splenic white cells and an increased proportion of cells with a large T-pattern, both data falling within normal limits. The effect of LVS appeared marked in thymectomized larvae, but was slight or absent in sham-operated larvae. Differential counts of the large T-pattern cells, based on the size of ANAE reaction product, showed the capacity of LVS to stimulate T-cell maturation. It was concluded that LVS induced proliferation and differentiation of splenic T-cells and that the different sizes of T-pattern reaction represent different maturational stages of T-cells.     

Increase in chemiluminescence induced by receptor-independent stimulation of polymorphonuclear cells from psoriatic patients

Using a lucigenin-dependent chemiluminescence the authors have measured the "respiratory burst" in polymorphonuclear cells from psoriatic patients and controls. Measurements were performed under stimulation with zymosan, phorbol 12-myristate 13-acetate and latex beads. It has been revealed that the response after stimulation with zymosan increased although there was no significant difference between patients and controls, while the response after stimulation with phorbol 12-myristate 13-acetate and latex beads was significantly increased. Our results suggest an enhanced metabolic activity of polymorphonuclear cells induced by stimuli acting independently from cell-membrane receptors. Therefore an enhanced excitability of polymorphonuclear leukocytes of psoriatic patients is supposed.     

大蒜不同品种蒜薹发育的解剖学研究

通过形态观测和石蜡切片法,比较了2个大蒜品种的蒜薹发育和解剖结构。结果表明：（1）“陇县火蒜”比“改良蒜”蒜薹的表皮细胞形状规则,排列致密;角质层较薄;（2）“陇县火蒜”比“改良蒜”蒜薹表面的气孔数量少,但开张度大;分泌细胞出现早、体积大、数量多;维管束数量少、直径小;（3）“陇县火蒜”蒜薹髓细胞卫多边形,髓细胞间隙率小,而“改良蒜”蒜薹的髓细胞呈椭圆形,髓细胞间隙率大。     

Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculenta Crantz) using computer image analysis

Tissue derived from embryogenic suspension cultures of cassava was bombarded with microparticles coated with a plasmid containing theuidA gene, which codes for-glucuronidase (GUS). After 3 days, the effect of different bombardment parameters was evaluated by comparing the numbers of blue spots that resulted from histological GUS assays. Counting of blue spots was performed using a system comprised of a black and white video camera, a stereoscope and a personal computer. A reproducible counting method was established by optimizing GUS assay conditions, preparation of tissue samples and acquisition of video images in view of attaining the highest possible contrast between the blue spots and the surrounding tissue. The effects of bombardment pressure, microparticle size, number of bombardments, and osmotic pretreatment on GUS expression were investigated. Optimal transient expression of theuidA gene was observed after bombardment at 1100 psi, with a particle size of 1 µm, an osmotic pretreatment and two bombardments per sample. The highest number of blue spots observed was 2400 per square centimeter of bombarded tissue.     

The Role of Lateral Blue Spots in Intrasexual Relationships Between Male Iberian Rock-Lizards, Lacerta monticola

Vertebrate males often show breeding colours that may function as reliable signals of status in intrasexual competition. In many lacertid lizards, males show a conspicuous row of small but distinctive blue spots that runs along their body side on the outer margin of the belly. However, no study has examined the role of these blue spots. We first analysed in a field population of the Iberian rock lizard, Lacerta monticola, the relationships between number of blue spots and some morphological traits, which are known to be related to males’ fighting ability. The number of spots seems to be an character showing ontogenetic change as large (generally older) males showed more blue spots than small (generally younger) males. Males with a higher body condition also showed a higher number of blue spots. Thus, a higher number of blue spots may be used to signal size, age or body condition. Many contiguous blue spots would result in a visual artefact consisting of a continuous blue band, which might be a reliable size‐ or condition‐dependent signal in some social contexts. We further examined in the laboratory whether male characteristics are related to dominance status. In males with similar body size or age, those with relatively larger heads were more dominant, whereas the number of blue spots was not important. Moreover, the number of blue spots in nature was not related to relative head size. Finally, we experimentally manipulated the presence and the number of blue spots of intruding males, and examined the aggressive response of resident males. Intruder individuals manipulated to cover all their blue spots received a lower amount of aggression. However, males with different numbers of manipulated blue spots received a similar number of aggressive responses. These results suggest that, during agonistic encounters, the presence of blue spots, but not their number, may elicit aggressiveness. Thus, blue spots may serve to identify an individual as an adult male, and to enhance body size of larger males.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

Defective B1 cell homing to the peritoneal cavity and preferential recruitment of B1 cells in the target organs in a murine model for systemic lupus erythematosus

We previously reported that B lymphocyte chemoattractant (BLC; CXCL13) was highly and ectopically expressed in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, and that B1 cells were preferentially chemoattracted toward BLC. We demonstrate in this study that B1 cells fail to home to the peritoneal cavity in aged BWF1 mice developing lupus nephritis, and that they are preferentially recruited to the target organs including the kidney, lung, and thymus when injected i.v. In contrast, B1 cells homed to the peritoneal cavity in aged BALB/c mice as effectively as in young mice. Accumulation of B1 cells to the omentum milky spots was also impaired in aged BWF1 mice compared with young mice. CD11bhighF4/80high cells with macrophage morphology were confirmed to be a major cell source for BLC in the peritoneal cavity both in young and aged BWF1 mice. However, the number of BLC-producing peritoneal macrophages was markedly decreased in aged BWF1 mice. These results suggest that the decreased number of BLC-producing peritoneal macrophages together with ectopic high expression of BLC in aged BWF1 mice result in abnormal B1 cell trafficking during the development of murine lupus.