Natural and inducible regulatory B cells are widely distributed in ovine lymphoid tissues.

Regulatory B cells that produce IL-10 are now recognized as an important component of the immune system. We previously confirmed that IL-10 secreting CD21+ regulatory B cells (Breg cells) were present in ovine jejunal Peyer's patches (JPP) and this IL-10 production suppressed IL-12 and IFN-γ secretion. It is not known, however, whether ovine Breg cells are restricted to JPP or are present in other lymphoid tissues. Therefore, CD21+ B cells were purified from sheep JPP and from a variety of mucosal and systemic lymphoid tissues using magnetic cell sorting. Purified CD21+ B cells were stimulated with a TLR9-agonist, CpG oligodeoxynucleotide (CpG ODN), and the frequency of spontaneous and inducible (i) IL-10-secreting B cells was evaluated by ELISPOT. Spontaneous IL-10 secreting CD21+ B cells were present in mucosal (jejunal PP, parabronchial lymph nodes (LN), mesesnteric LN, and palatine tonsils) and systemic (spleen and blood) lymphoid tissues. Mucosal lymphoid tissues (parabronchial and mesenteric LNs and JPP) had the highest frequency of cells spontaneously secreting IL-10 while tonsils had the lowest. The frequency of B cells spontaneously secreting IL-10 was lowest in blood and spleen. There was large inter-animal variation in the frequency of CD21+ B cells spontaneously secreting IL-10 and no significant difference was detected following CpG ODN stimulation. When comparing within individual animals there was, however, a consistent increase in the frequency of CD21+ cells secreting IL-10 following CpG ODN stimulation versus stimulation with GpC control ODN. The presence of inducible (i)Breg cells in ovine mucosal tissues supports previous evidence from mice indicating that B cells have the capacity to modulate inflammatory responses. The presence of iBreg cells in ruminants may also provide a novel therapeutic target for both immunomodulatory drugs and vaccines designed to control antigen-specific mucosal inflammation.

Evidence for the existence of regulatory and effector B cell populations in Peyer's patches of sheep.

IL-10 secreting CD21(+) B cells exist in sheep Peyer's patches (PP). It's not known however, whether all PP B cells are regulatory or whether an effector population also exists in this tissue. To further characterize the subpopulations of B cells in PP's, highly purified B cells were negatively sorted from jejunal PP and fractionated according to co-expression of CD72(+)CD21(+)or CD72(+)CD21(-) molecules and then stimulated with the TLR9-agonist, CpG ODN. IL-10, IL-12, IFN-γ, and IgM production were then assayed. We observed that only highly purified CD72(+)CD21(+) B cells spontaneously secreted high levels of IL-10, but they did not produce any IL-12, IFN-γ or IgM suggesting that this cell population contains regulatory B cells. In contrast, CD72(+)CD21(-) B cells did not secrete IL-10, but secreted IL-12, IFN-γ, and IgM, suggesting they include effector cells. In addition, B cells expressing surface IgA, IgM and IgG1 all secreted similar levels of IL-10. We further confirmed that only B cells produce IL-10, while other cells in the PP including DCs and T cells do not. Our investigations may provide evidence for the existence of two sub-populations in sheep PP; IL-10 secreting regulatory (CD72(+)CD21(+)) cells, and IL-12/IFN-γ/IgM-secreting effector (CD72(+)CD21(-)) cells.

Attenuation of pain behaviour by local administration of alpha-2 adrenoceptor antagonists to dorsal root ganglia in a rat radiculopathy model.

BACKGROUND: There were several reports suggesting α-adrenoceptor antagonists are effective to treat neuropathic pain. The aims of this study were as follows: (1) to introduce drug delivery system for dorsal root ganglion (DRG) neurons; (2) to elucidate the effects of α-adrenoceptor antagonists in acute, subacute or chronic phase and (3) to determine which subtype of adrenoceptor was mainly involved. METHOD: We used 130 male Sprague-Dawley rats. After root constriction, rats received three local injections of α-adrenoceptor antagonists around DRG. We administered the non-selective α-adrenoceptor antagonist phentolamine for 3 consecutive days from day 0, 4 or 11 after the surgery, and the α1-adrenoceptor antagonist prazosin, the α1-adrenoceptor antagonist silodosin, the more preferred α1-adrenoceptor than prazosin and the α2-adrenoceptor antagonist yohimbine for 3 consecutive days from day 0 after the surgery. RESULTS: Phentolamine and yohimbine continually attenuated pain behaviour. Prazosin at high dose attenuated pain behaviour, however, prazosin at low dose did not attenuate pain behaviour every experimental day. Silodosin had no analgesic effect. Phentolamine injections from day 4 after surgery attenuated pain behaviour that had been established on the 3rd experimental day until the 28th post-operative day, although effect of phentolamine wore off. Phentolamine injections from day 11 after surgery temporarily attenuated pain behaviour that had been established on the 3rd, 7th and 10th experimental days. CONCLUSIONS: This study showed α-adrenoceptor antagonists could suppress pain behaviour via α2-adrenoceptor in acute phase and temporary attenuate pain behaviour in chronic phase. These findings presented potentials sympathetic nerve blockade contributed to treat neuropathic pain.

IL-10 secreting CD21⁺ B cells are present in jejunal Peyer's patches of sheep during fetal development.

We recently reported a novel interleukin-10 (IL-10)-secreting CD21(+) B cell population in jejunal Peyer's patches (JPP) of sheep with a regulatory function (Bregs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21(+) B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21(+) B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for Bregs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21(+) B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21(+) B cells in PPs prior to antigen exposure.

[The possibility of acute inflammatory reaction affects the development of pressure ulcers in bedridden elderly patients].

To test the hypothesis that acute inflammatory reaction associates with the development of pressure ulcers in bedridden elderly patients, 40 hospitalized elderly patients suffering from bacterial pneumonia, cerebrovascular disease, and femoral bone fracture were enrolled in this study. All of them were divided into two groups with pressure ulcers (group P; 17 patients) and without one (group N; 23 patients). The blood samples were taken from them within 5 days after the patients being bedridden. Although no significant difference exist in pressure ulcer risk factors (age, gender, Braden scale, underlying diseases, blood pressure, and heart rate) between the two groups, white blood cell, plasma C-reactive protein and fibrinogen in group P increased significantly as compared with those in group N. Besides number of platelets and maximum platelet aggregation rate were significantly higher in group P than in group N. Serum albumin and hemoglobin of both groups decreased after being bedridden, especially hemoglobin in group P was significantly lower than that in group N. While the concentration of serum IL-6 did not indicate a significant difference between both the groups, serum IL-1 beta increased significantly in group P. In conclusion, we suggested that acute inflammatory reaction releasing proinflammatory cytokines affected the development of pressure ulcer in bedridden elderly patients.

[Quantitation and heterogeneity of serum alpha 2-macroglobulin].

alpha 2-Macroglobulin (alpha 2M) is a major protease inhibitor in human plasma. In this study, serum alpha 2M was determined by means of immunoelectrophoresis (IEP), rocket immunoelectrophoresis (R-IEP), laser nephelometry (LN) and single radial immunodiffusion (SRID) in 133 subjects. The values obtained by these methods coincided well in most specimens, but some specimens showed disagreement. The sera of alpha 2M deficiency found by IEP were analyzed by the other methods. We found that R-IEP gave the results of alpha 2M deficiency most frequently while the other methods gave the results of normal alpha 2M level for some specimens. We studied on the presumption that the phenomenon is due to heterogeneity in molecular weight of alpha 2M. After alpha 2M was purified by affinity chromatography and gel filtration chromatography, we further employed gel-filtration on a high-performance liquid chromatography (HPLC) to isolate alpha 2M with different molecular size. Two peaks of immunoreactive alpha 2M were found by the gel-filtration HPLC. When we performed IEP for both portions, the first portion with higher molecular size showed larger rate of migration than the second portion. Serum alpha 2M quantitation with SRID is not appropriate because molecular weight influences on the determination by SRID. We conclude that R-IEP is best suitable for the detection of decrease of serum alpha 2M, although this method requires expertness of manipulation.

[Concentrations of free form and complex form of prostate-specific antigen in serum of alpha 2 macroglobulin deficient patients with prostatic carcinoma].

Prostate-specific antigen (PSA) is present in blood in free form as well as in complex form with various protease inhibitors such as alpha 2 macroglobulin (alpha 2M) and alpha 1 antichymotrypsin (alpha 1 ACT). We had found that alpha 2M is deficient (below approximately 40 mg/dl) in some patients with prostatic carcinoma. Therefore, we investigated the levels of free and complex form of PSA in patients with prostatic disease and obtained the following results. The HPLC study showed that total (free plus complex) PSA level was much higher in the alpha 2M deficient patients with prostatic carcinoma stage D (n = 7, range 1,530-14,746 ng/ml, median value 6,800 ng/ml) than in the non-deficient patients with stage D (n = 16, range 121.6-4,210 ng/ml, median value 851 ng/ml). In the deficient patients, the complex of PSA with alpha 1 ACT increased extraordinarily while free PSA increased to only some extent. In the more severe cases of prostatic carcinoma, the ratio (complex/total PSA) was higher while the ratio (free/total PSA) was lower. The SDS-PAGE Western blotting showed that complex PSA increased extraordinarily and free PSA increased in sera of the deficient patients which was consistent with the HPLC results. Many bands appeared on the blotting at the positions smaller than alpha 2M molecule, which indicated that many fragments of alpha 2M were present in their sera. These bands were more intense than the bands with sera of controls or benign prostatic hypertrophy. The alpha 2M deficiency may be due to the rapid disappearance of the complex with PSA or any other prostate-originated proteases. The complex may undergo accelerated degradation or catabolism of alpha 2M.

[Clinical study on prostate cancer initially presenting with disseminated intravascular coagulation syndrome].

We experienced five patients with prostate cancer with disseminated intravascular coagulation syndrome (DIC) at the first presentation at Gunma University Hospital and affiliated institutions between 1991 and 1997. Their average age was 68 years, average DIC score at the first presentation was 10 and prostate specific antigen (PSA) level was more than 700 ng/ml. All of them had multiple bone metastases. The therapy for DIC and hormonal therapy for prostate cancer were simultaneously started at the first presentation before prostate needle biopsy, but all patients died. The average number of days from the start of DIC to death was 685 days. The patients initially showed a good response to therapy, but their conditions soon aggravated. The prognosis was extremely poor, but some proper therapies lead to the prognosis which was equal to that of prostate cancer in Stage D2 without DIC.

[A study of the association between HLA phenotype and serum concentration of soluble HLA class I].

We investigated the association between the phenotypes of human leucocyte antigens (HLA) class I on the surface of lymphocytes and serum concentrations of soluble HLA (sHLA) in normal and Human immunodeficiency virus (HIV) infected subjects. Serum concentrations of sHLA inn normal subjects with HLA-A 24 were significantly higher than those in such subject without HLA-A 24. The similar relation was found in HIV infected subjects whose levels of sHLA significantly increased compared with that of normal subjects. These results might suggest that the mechanism which causes the increase in secretion of sHLA in HIV infected subjects does not change the association between HLA phenotype and serum concentration of sHLA.

The Relation between Superoxide Dismutase in Cancer Tissue and Clinico-pathological Features in Breast Cancer.

The localization of Cu/Zn- and Mn-superoxide dismutase (SOD) in breast cancer tissue (12 papillotubular carcinomas, 21 solid-tubular carcinomas, 16 scirrhous carcinomas, 1 medullary carcinoma, 1 secreting carcinoma, 1 lobular carcinoma, 1 Paget's disease) was investigated via an immunohistochemical technique using antihuman Cu/Zn- and Mn-SOD antibodies in 10%formalin fixed-paraffin embedded thin sections. Both SODs stained strongly in the normal breast gland, but not clearly in many cancer tissues. Furthermore, Cu/Zn-SOD stained more strongly in well differentiated tubular carcinomas than in poorly differentiated tubular carcinomas. It tended to stain less in tumors which recurred or had a poor outcome, and in tumors with a diploid pattern on DNA flow cytometry. Mn-SOD staining was similar to that of Cu/Zn-SOD, but no significant differences among subgroups was found, since the incidence of positively staining tumors was too small in all groups. The intensity of SOD staining seems to change in relation to cell proliferation and differentiation in breast carcinoma, and may be a prognostic indicator, since SOD decreased in poorly differentiated carcinoma and in tumors which developed distant metastasis. Thus, the localization of SOD in breast cancer tissue can provide useful information for cancer treatment.

Localization of Cu/Zn and Mn superoxide dismutase in various thyroid disorders.

The intracellular localization of Cu/Zn- and Mn-superoxide dismutase (SOD), which catalyze the dismutation of superoxide radicals (O2-) to O2 and H2O2, was studied in the thyroid tissue of various thyroid disorders by an immunohistochemical technique. The concentrations of both SODs in those tissues were measured also by a sandwich enzyme immunoassay technique. Copper/zinc-SOD in thyroid tissues were identified by immunocytochemical staining in most cases of papillary carcinoma and in some cases of other thyroid disorders. In normal follicular cells this enzyme is localized in the perinuclear cytoplasm, whereas in thyroid tumor or hyperplastic follicular cells it exists homogeneously in cytoplasm. Manganese-SOD stained strongly in papillary carcinoma and papillary-growing cells in the thyroid tissue of adenoma and Graves' disease. The concentrations of Cu/Zn-and Mn-SOD in thyroid tumor tissues and hyperplastic follicular disorders were significantly higher than those in normal thyroid tissue when they were compared as a function of protein or deoxyribonucleic acid contents. The ratio of Mn-SOD to Cu/Zn-SOD was significantly higher only in papillary carcinoma, except for other thyroid disorders as compared with that in the normal thyroid. In conclusion, SOD seems to be related to cell proliferation and differentiation in the thyroid follicular cell because Cu/Zn-SOD changes its localization in tumor and hyperplastic follicular cells and because the Mn-SOD concentration is increased in papillary carcinoma or papillary-growing cells.

Polymorphonuclear leucocyte function tests: a comparison of cytochrome C reduction and flow cytometric analysis.

Assay results were compared between the cytochrome C reduction test and flow cytometry using fluorescent beads or DCFH-DA (2', 7'-dichlorofluorescein diacetate) granules, in examining the phagocytosing and killing properties of polymorphonuclear leucocytes (PMN). When PMN samples from 20 healthy persons were assayed, no correlation was found between the cytochrome test and the phagocytosis (fluorescent beads) test or phagocytosing-killing (DCFH-DA) test by flow cytometry techniques. It is suggested that this might be caused by the fact that for technical reasons the two tests employed different stimulants and different substances to be phagocytosed. The simple flow cytometry procedure is now in widespread use as a PMN function test rather than the cytochrome C or nitroblue tetrazolium (NBT) dye reduction tests, and caution should be exercised in comparing and interpreting test results reported from separate laboratories. Because results using these two tests do not necessarily agree with each other, it seems preferable for PMN function testing to employ a combination of two or more approaches relying on different assay principles.

[Prostatic specific antigen (PA) in mass screening for prostate cancer].

The usefulness of prostatic specific antigen (PA) was compared with that of prostatic acid phosphatase (PAP). PA was determined in the serum of 2,183 patient examined by the mass screening for prostate cancer from 1987 to 1990. The serum samples of these patients were obtained from our serum bank. PA was measured by the E test "TOSOH" II (PA). The relationship of PA and PAP to prostate size estimated by digital rectal examination (DRE) and ultrasound tomography (US), and age was investigated. PA and PAP correlated with aging and prostate size estimated by DRE. However PA was more apparently related with these things. The correlation between PA and prostatic size estimated by US was relatively high (r = 0.53), but the correlation between PAP and prostate size estimated by US was low (r = 0.20). When the upper limit of normal range was set at 6.0 ng/ml, the sensitivity, specificity and efficiency was 64%, 97% and 62%, respectively. PA was more sensitive than PAP and could be more useful since none of the patients with prostate cancer was PAP positive and PA negative. We conclude that PA should be a reliable tumor marker in our mass screening system.

Plasma catecholamines determination using high pressure liquid chromatography and their roles in blood pressure regulation and experimental hypertension in rats.

Plasma catecholamine levels have been used experiemtally and clinically as the indices of the sympathetic nerve activity. We measured plasma catecholamines using high pressure liquid chromatography in rats to assess the significance of plasma catecholamines as an index of the sympathetic nerve activity and its role in hypertension. Pentobarbital anesthesia depressed plasma catecholamine levels, especially plasma adrenaline. Sodium loading for 5 weeks suppressed plasma noradrenaline, while administration of furosemide (1 mg/kg) produced the elevation of plasma noradrenaline. Experimental hypertension, one-kidney and two-kidney types of Goldblatt hypertension and DOCA-salt hypertension, raised plasma noradrenalines both in acute and chronic phases. The infusion of pressor doses of angiotensin II suppressed plasma noradrenaline by the reflex mechanism. Sar1, Ile8-angiotensin II and SQ 14,225 did not suppress plasma cathecholamine elevation due to hemorrhage. L-Hydroxyldopamine produced elevation of plasma catecholamines in experimental nypertension and controls in rats. After adrenal demedullation, plasma noradrenaline was decreased by the administration of 6-hydroxy-dopamine. Acute reduction of circulating blood volume and blood pressure fall produced the elevation of plasma catecholamine, especially plasma adrenaline. In rats, the adrenal medulla plays an important role in the regulation of blood pressure.