Pathological changes of adipose tissue in secondary lymphoedema.

BACKGROUND: The pathophysiology of lymphoedema is poorly understood. Current treatment options include compression therapy, resection, liposuction and lymphatic microsurgery, but determining the optimal treatment approach for each patient remains challenging. OBJECTIVES: We characterized skin and adipose tissue alterations in the setting of secondary lymphoedema. METHODS: Morphological and histopathological evaluations were conducted for 70 specimens collected from 26 female patients with lower-extremity secondary lymphoedema following surgical intervention for gynaecological cancers. Indocyanine green lymphography was performed for each patient to assess lymphoedema severity. RESULTS: Macroscopic and ultrasound findings revealed that lymphoedema adipose tissue had larger lobules of adipose tissue, with these lobules surrounded by thick collagen fibres and interstitial lymphatic fluid. In lymphoedema specimens, adipocytes displayed hypertrophic changes and more collagen fibre deposits when examined using electron microscopy, whole-mount staining and immunohistochemistry. The number of capillary lymphatic channels was also found to be increased in the dermis of lymphoedema limbs. Crown-like structures (dead adipocytes surrounded by M1 macrophages) were less frequently seen in lymphoedema samples. Flow cytometry revealed that, among the cellular components of adipose tissue, adipose-derived stem/stromal cells and M2 macrophages were decreased in number in lymphoedema adipose tissue compared with normal controls. CONCLUSIONS: These findings suggest that long-term lymphatic volume overload can induce chronic tissue inflammation, progressive fibrosis, impaired homeostasis, altered remodelling of adipose tissue, impaired regenerative capacity and immunological dysfunction. Further elucidation of the pathophysiological mechanisms underlying lymphoedema will lead to more reliable therapeutic strategies.

Multiple ulcers in the small and large intestines occurred during tocilizumab therapy for rheumatoid arthritis.

Tocilizumab is a monoclonal antibody against human interleukin-6 receptor which blocks the binding of interleukin-6 to its receptor. Tocilizumab is effective for the treatment of inflammatory disorders including rheumatoid arthritis. We report a case of multiple ulcers in the small and large intestines, which occurred during tocilizumab therapy. A 57-year-old woman started to use tocilizumab for rheumatoid arthritis. Three months later, she complained of hematochezia. Double-balloon endoscopy revealed multiple small aphthoid ulcers in the small and large intestines. One month after the woman had recovered, she was given tocilizumab again. The woman had hematochezia and abdominal pain again 2 weeks later. Colonoscopy revealed multiple round, discrete punched-out ulcers in the terminal ileum, and vast deep ulcers from the cecum to the descending colon. Bioptic histopathology and cultivation showed non-specific findings. Six weeks after discontinuation of tocilizumab, ulcers in the small and large intestine dramatically improved, leaving ulcer scars. This disease course and the results of examination made us strongly suspect that tocilizumab induced multiple ulcers in the small and large intestines. Interleukin-6 is a pleiotropic cytokine and involved in intestinal mucosal wound healing as well as in inflammatory processes. It is possible that tocilizumab inhibited tissue repair of the intestine and caused intestinal ulcers.

Impact of double-balloon endoscopy on the diagnosis of jejunoileal involvement in primary intestinal follicular lymphomas: a case series.

In recent years, primary gastrointestinal follicular lymphoma has been increasingly detected in the duodenum on esophagogastroduodenoscopy (EGD). Primary gastrointestinal follicular lymphomas are frequently distributed to multiple sites in the gastrointestinal tract. Therefore, investigation into the spread of follicular lymphomas in the small bowel is important in order to determine the most appropriate treatment strategy. The performance of double-balloon endoscopy (DBE) in the diagnosis of jejunoileal follicular lymphoma lesions has not been fully evaluated. We aimed to investigate the value of DBE in addition to computed tomography (CT) and (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG-PET) in the diagnosis of jejunoileal follicular lymphoma. DBE with biopsy was performed in seven patients with primary duodenal follicular lymphoma diagnosed by EGD, in order to investigate jejunoileal involvement. Jejunoileal follicular lymphoma lesions were detected by DBE in six out of the seven patients (three in the jejunum and three in the jejunum and ileum), whereas CT and (18)F-FDG-PET failed to detect the existence of these lesions. Endoscopic findings of the jejunoileal lesions revealed multiple white nodules and white villi, which were similar to those of duodenal lesions. DBE was more useful for the diagnosis of jejunoileal involvement in primary intestinal follicular lymphoma than CT and (18)F-FDG-PET. The use of DBE will become important for determining the most appropriate treatment for gastrointestinal follicular lymphoma.

Negative MRI findings in a case of degenerative myelopathy in a dog.

An 11-year-old male Rough collie was submitted with paraparesis, but did not respond to medical treatment. Clinical signs worsened and the dog displayed paralysis, inability to stand and loss of voluntary bladder control, whereupon magnetic resonance imaging (MRI) was performed. No significant abnormalities were identified from MRI, blood tests, cerebrospinal fluid tests or radiography. After MRI, the dog developed dyspnoea and died. Autopsy and subsequent histopathological examination led to a diagnosis of degenerative myelopathy.

Preparation of functionally preserved CD4+ CD25high regulatory T cells from leukapheresis products from ulcerative colitis patients, applicable to regulatory T-cell transfer therapy.

BACKGROUND: Ulcerative colitis (UC) is an intractable disease; therefore new therapies need to be developed. CD4(+) CD25(high) regulatory T cells (Treg) significantly ameliorate colitis in animal models. In active UC patients, although Treg are functionally preserved, their proportion in peripheral blood decreases. Thus Treg transfer therapy is expected to be efficacious for UC. During leukapheresis for UC, Treg are depleted, as well as colitogenic effector leukocytes. We therefore designed a leukapheresis/Treg transfer therapy in which Treg are isolated from leukapheresis products and transfused to patients, and studied large-scale germ-free methods of Treg preparation. METHODS: Using the CliniMACS cell selection system, we conducted Treg isolation experiments from leukapheresis products in which B and CD8(+) T cells were depleted, followed by positive selection of CD25(+) cells. In some experiments, isolated Treg or non-Treg were expanded with interleukin-2 (IL-2) +/- transforming growth factor (TGF)-beta1. Expression of a Treg-specific marker, FOXP3, and gut-homing receptors, and suppressor activity of isolated or cultured cells, were analyzed. RESULTS: CD4(+) CD25(high) T cells were collected and efficiently enriched with a good recovery rate. Isolated cells preferentially expressed FOXP3 and significantly suppressed T-cell proliferation in vitro. In addition, isolated Treg could be efficiently expanded, and Treg could be induced from non-Treg with TGF-beta1 in vitro. TGF-beta1 significantly up-regulated alphaEbeta7 and alpha4beta7 integrins. DISCUSSION: We have established a method of Treg isolation from leukapheresis products that can be used clinically; therefore, Treg transfer therapy is feasible in combination with leukapheresis for UC. Expansion or induction of Treg in vitro may be another approach to Treg-based immunotherapy.

Clinical improvement in two dogs with hydrocephalus and syringohydromyelia after ventriculoperitoneal shunting.

Two young Miniature Dachshunds were presented with abnormal gait. Magnetic resonance imaging showed, hydrocephalus with expanding fourth ventricle, and syringohydromyelia in the cervical spinal cord. These dogs underwent ventricle-peritoneal shunting, after which hydrocephalus, syringohydromyelia, and their clinical signs, improved.

MRI findings of haematomyelia in a dog with spontaneous systemic haemorrhage.

A 7-year-old female cross-breed dog was brought to Nihon University Animal Medical Center for investigation of tetraplegia. Lameness in the pelvic limbs, that had developed 2 weeks previously, had progressed to tetraplegia. On magnetic resonance imaging of the spinal cord, isointensity was detected from C2 to C4 and T12 to T13, isointensity and hyperintensity were intermingled from L3 to L4, and hyperintensity was detected from L5 to L7 by T1-weighted imaging. On T2-weighted imaging, hyperintensity was detected in all regions described above. The dog recovered from anaesthesia, but died during the day from systemic bleeding as the result of a coagulopathy of unknown aetiology. Histopathological examination revealed haematomyelia in these regions of the spinal cord. This is the first report of magnetic resonance imaging findings of haematomyelia in canine spontaneous systemic haemorrhage. It appeared that the differences in the findings of T1-weighted imaging along the spinal regions reflected time-lags in the occurrence of bleeding.

The clinical significance of cerebrospinal fluid lipid peroxides in central nervous system disease.

Forty-four dogs were referred to our hospital presenting with neurological symptoms such as seizure or paraparesis. Magnetic resonance imaging (MRI) revealed abnormal results in 21 (abnormal MRI group) and normal results in 23 dogs (normal MRI group). Cerebrospinal fluid (CSF) (normal MRI group, n = 22; abnormal MRI group, n = 21) and serum lipid peroxide (LP) concentrations (normal MRI group, n = 11; abnormal MRI group, n = 15) were measured in a number of these dogs, and revealed a significant difference in the CSF/serum LP values (normal MRI group, n = 10; abnormal MRI group, n = 14) between the abnormal and the normal MRI groups (t-test and Mann-Whitney U-test p < 0.05). No other significant differences were observed. CSF/serum LP values exceeding 1.0 were exhibited in 10 of 14 dogs (71%) in the abnormal MRI group, and in 1 of 10 dogs (10%) in the normal MRI group. In the remaining animals, 4 dogs of the abnormal MRI group showed CSF/serum values lower than 1.0, 3 dogs had morphological abnormalities but no abnormal MRI signals in the central nervous system, and 1 dog had an abnormal MRI signal but no pathological abnormality. In the CSF analysis, 3 of 16 dogs (19%) of the abnormal MRI groupshowed abnormal cell counts and/or protein content. We conclude that the CSF/serum LP value can be used for the detection of neurological lesions such as oedema, inflammation and tumour.

Subdural accumulation of fluid in a dog after the insertion of a ventriculoperitoneal shunt.

A dog with hydrocephalus as a result of aqueduct stenosis and cerebellar herniation underwent ventriculoperitoneal shunting. Magnetic resonance images and computed tomography scans taken after the surgery revealed subdural accumulations of haemorrhagic fluid and cereberocortical collapse caused by overshunting and leakage of cerebrospinal fluid from the site of insertion of the shunt. However, the degree of cerebellar herniation was reduced after the shunt was inserted, and the dog did not develop any neurological signs and made good progress.

Purification of protein phosphatase from hen oviduct.

Two protein phosphatases of 103 and 29 kDa as determined by gel filtration, were purified from hen oviducts. The 103 -kDa phosphatase was purified 7300-fold to near homogeneity and dissociated into two polypeptides in the presence of SDS. Molecular masses of these polypeptides were estimated to be 60 and 38 kDa by SDS-polyacrylamide slab gel electrophoresis using the buffer system of Laemmli, but 68 and 35 kDa using the buffer system of Weber and Osborn. The stoichiometry of these polypeptides was approx 1:1 according to the densitometric analysis of gels at 550 nm. The 29 -kDa phosphatase was purified 2900-fold. Both phosphatases dephosphorylated the alpha-subunit of phosphorylase kinase more rapidly than the beta-subunit.

Immunohistochemical study of gamma delta T cell receptor-positive cells in the capsular region of hepatocellular carcinoma: possible role in defense against expansion of carcinoma in the liver.

The localization and distribution of gamma delta T cell receptor (TCR)-positive cells (gamma delta T cells) in hepatocellular carcinoma capsules was investigated immunohistochemically at both light and electron microscopic levels. Most of the mononuclear cells infiltrating the tumor capsules were CD3-positive. Together with gamma delta T cells, they were significantly increased in the tumor capsules compared to amounts in the fibrous septa in non-cancerous cirrhotic areas of the same liver, and compared to amounts in the liver of patients with cirrhosis. Phenotypic characterization by the two-color double-staining technique showed that CD8/gamma delta cells were significantly increased in the tumor capsule, and that more than one-third of gamma delta TCR-positive cells also expressed the CD56 antigen. Morphological observation revealed that large gamma delta T cells were increased in number in the tumor capsule and that the cytoplasm of these cells contained multivesicular bodies and dense granules. These morphological features were similar to those of large granular lymphocytes, and most of the gamma delta T cells were also positive for BB3. This suggests that extrathymic maturation of gamma delta T cells occurs in the tumor capsule, and that these gamma delta T cells may have a cytolytic effect on tumor cells, as shown in large granular lymphocytes; further, the results suggest that these cells may play a role in the defense against tumor expansion.

Expression of adhesion molecules and HLA-DR by macrophages and dendritic cells in aphthoid lesions of Crohn's disease: an immunocytochemical study.

The phenotypes and ultrastructure of macrophages and dendritic cells in aphthoid lesions of the colon were immunocytochemically observed in patients with Crohn's disease. Biopsy specimens were endoscopically obtained from both aphthoid and advanced lesions in Crohn's disease patients. Biopsy specimens obtained from patients with infectious colitis and from normal individuals served as controls. Aphthoid lesions contained densely aggregated CD68+ macrophages, which were surrounded by numerous ID-1+ dendritic cells. In the normal controls and infectious colitis patients, however, a few scattered CD68+ macrophages and ID-1+ dendritic cells were noted beneath the surface epithelium. CD3+ lymphocytes were significantly increased in both aphthoid and advanced lesions of Crohn's disease, but the CD4/CD8 ratio was similar in all groups studied. The double immunoperoxidase staining method revealed that both CD68+ macrophages and ID-1+ dendritic cells in the aphthoid lesions simultaneously expressed ICAM-1 and HLA-DR antigens. Electronmicroscopic observation revealed that CD68+ macrophages had numerous vesicles and lysosomal granules and few projections, and that ID-1+ dendritic cells had appreciable cytoplasmic protrusions with a few vacuoles. These findings suggested that the colonic mucosa in Crohn's disease contained two types of macrophage/dendritic cells in the same lineage that expressed intercellular adhesion molecules and class-II MHC antigens. It also appeared that the aphthoid lesions of Crohn's disease featured an increase in macrophages and dendritic cells consistent with immunological activation.

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.

Relationship between changes in volume of the oviductal fluid in the ampulla and the descent of ovulated eggs from the ampulla to the isthmus in mice.

Follicular growth and ovulation were induced in mice by administration of pregnant mare serum gonadotrophin (PMSG) followed 2 days later by human chorionic gonadotrophin; the day of PMSG injection was designated as day 0. The volume of the ampulla was measured and the location of the ovulated eggs determined at 06:00, 10:00, 14:00, 18:00 and 22:00 on day 3, and at 02:00 and 06:00 on day 4. The volume of the ampulla and hence oviductal fluid, peaked at 14:00 on day 3 and then declined. In all oviduct samples taken up to 14:00 on day 3, eggs were found exclusively in the ampulla. Thereafter, an increasing number of eggs were observed in the isthmus. Thus, the migration of eggs from the ampulla to the isthmus was concurrent with the decrease in oviductal fluid volume. The peak in the volume of oviductal fluid seen at day 3 is likely to coincide with the opening of the ampullary-isthmic junction of the oviduct.

The yield of abnormal unfertilized eggs observed after repeated gonadotrophin-induced ovulation.

This study investigated whether or not the yield of abnormal eggs produced after repeatedly induced ovulation could be reduced by increasing the interval between gonadotrophin (GTH) administrations. Ovulation was induced, in a mouse model, by subcutaneous injection of pregnant mare serum GTH and human chorionic GTH given once (n = 10), twice (n = 10), three times (n = 10) or four times (n = 10) at intervals of 5 days. In 30 further mice, a fifth induction was performed either 5 (n = 10), 10 (n = 10) or 20 days (n = 10) following the fourth injection. Oviducts were examined microscopically, and the eggs were counted and examined for morphological abnormalities. In the mice who had received four GTH treatments, 33.3% (12 of 36) of the eggs were abnormal. After the fifth induced ovulation performed after an interval of 5, 10 or 20 days, the yield of abnormal eggs was 50.0% (five of 10), 23.1% (nine of 39) and 15.2% (seven of 46), respectively. These results indicate that prolongation of the interval between GTH administrations reduced the yield of abnormal eggs produced following repeatedly induced ovulation.

Effects of splenectomy on luteal function in pseudopregnant rabbits.

The involvement of the immune system in changes in luteal function was evaluated in rabbits. Pseudopregnancy was induced in 10 females and the spleens (considered to be the source of macrophages) of five were excised on day 7 of pseudopregnancy, while the five controls had sham splenectomies. Subsequent changes in serum progesterone concentrations were measured as an indicator of luteal function and luteolysis. A second pseudopregnancy was induced 31 days after splenectomy. The first pseudopregnancy was prolonged and during the second pseudopregnancy the serum progesterone concentrations on days 3 and 7 were much lower in the splenectomy group than in the control group. On day 14 of the second pseudopregnancy, the serum progesterone concentration markedly decreased in the control group while it remained almost at the level of day 7 in the splenectomy group. These results suggest that splenectomy suppresses the expression of luteal function and delays luteolysis in rabbits.

Phagocytotic activity of macrophages separated from corpus lutea of pseudopregnant rabbits.

Macrophage activity as indicated by phagocytosis in the corpus luteum at the luteolysis stage was examined in rabbits during pseudopregnancy. The day of induction of pseudopregnancy was regarded as day 0, and the phagocytotic activity of macrophages obtained from luteal tissues was examined on days 7 and 16. When sheep red blood cells (SRBCs) were used to assess phagocytotic activity there was no difference in phagocytosis between days 7 and 16 but, when sensitized SRBCs were used to assess phagocytotic activity, the activity of macrophages on day 16 was more than double that on day 7 (P < 0.05). These results indicate that immunomediated phagocytosis of luteal macrophages is enhanced at the luteolysis stage (day 16). This phenomenon may contribute to the process of luteolysis.

Cleavage of unfertilized eggs after repeated administration of human chorionic gonadotrophin to hamsters.

The proportions of unfertilized eggs in the oviducts, showing abnormal cleavage, were examined in hamsters given single or repeated doses of 30 i.u. human gonadotrophic hormone for the induction of ovulation. In control animals (n = 7), 1.7% of the total ovulated eggs were morphologically abnormal unfertilized eggs showing cleavage. The proportions of unfertilized eggs that were abnormal in the groups of seven hamsters treated with one, two or three doses of the gonadotrophin were 20.4%, 19.4%, and 30.4%, respectively. The proportion of unfertilized eggs showing abnormal cleavage thus appeared to increase with repeated administrations of gonadotrophin.