Coronary artery complicated lesion area is related to functional polymorphism of matrix metalloproteinase 9 gene: an autopsy study.

Matrix metalloproteinase 9 (MMP9) is expressed in human atherosclerotic plaques, and the protein is localized in human coronary atherosclerotic lesions. The MMP9 gene has a C-to-T promoter polymorphism at position -1562, which affects transcription and leads to promoter low-activity (C/C) and high-activity (C/T, T/T) genotypes. To determine whether these genotypes exert an influence on the atherosclerotic lesion area, we investigated their association with different types of coronary lesions in an autopsy cohort of 276 men aged 33 to 69 years. Areas of the coronary wall covered with fatty streaks and fibrotic, calcified, and complicated lesions were measured, and the percentage of coronary narrowing was determined. MMP9 genotypes were determined by polymerase chain reaction and restriction enzyme digestion. In men aged >/=53 years, the mean area of complicated lesions in 3 coronaries was significantly associated with the MMP9 genotype (P=0.008). Subjects with high promoter activity genotypes had, on average, larger complicated lesion areas than did those with the low-activity genotype. The MMP9 genotype persisted as an independent predictor of complicated lesion area after adjustment for age, body mass index, hypertension, diabetes, and smoking (P=0.012). These data provide evidence that the proposed effect of MMP9 in the process of atherosclerotic lesion development may be modified by the MMP9 genotype.

Reactions of Leydig cells and blood vessels to lymphoblastic leukemia in the rat testis.

The testis is the third common site of relapse in childhood acute lymphoblastic leukemia (ALL). Despite the apparent clinical importance of testicular relapse, its pathogenesis is still unknown. The studies with an animal model of ALL have shown that many testicular factors are able to control the intratesticular infiltration and proliferation of leukemic lymphoblasts during the untreated course of ALL. In the present study, the ultrastructure of rat testicular interstitium infiltrated by leukemic lymphoblasts was studied in two groups of rats transplanted with rat T cell leukemia in early and late puberty. In both groups most of the leukemic cells infiltrating testicular interstitium were totally or partly enveloped by one or more Leydig cells, and the endothelial cells of capillaries, arterioles and venules were hypertrophic. The Leydig cells of the younger experimental group were by nuclear and cytoplasmic ultrastructure similar to the undifferentiated Leydig cells normally seen on the third postnatal week. The results suggest that Leydig cells bind leukemic lymphoblasts on their surface in vivo as also previously observed in vitro, and that ALL may disturb the pubertal maturation of Leydig cells. The occlusion of arterial and capillary lumina by folds of hypertrophic endothelial cells together with adhered leukemic lymphoblasts may impair the circulation of leukemic testes.

Regulation of testicular infiltration in acute lymphoblastic leukaemia of the rat.

The testis is a common site of relapse in childhood acute lymphoblastic leukaemia (ALL). In adults, testicular relapses of ALL are very rare. A similar age-difference in the frequency of the testicular infiltration exists also in the rat T-cell leukaemia. In the present investigation, the effect of various hormonal treatments and unilateral cryptorchidism on the form of testicular infiltrates by the rat T-cell leukaemia was studied. Inhibition of testicular activity by estradiol treatment (E2) of early pubertal rats injected i.p. with rat T-leukaemic lymphoblasts significantly decreased the proportion of the testis occupied by leukaemic infiltrates. The proportion of the testis occupied by leukaemic infiltrates was significantly higher in the abdominal testes of both early and late pubertal unilaterally cryptorchid rats, than in the scrotal testes of leukaemic control rats. Daily treatment of early pubertal rats with human chorionic gonadotrophin (hCG), or human menopausal gonadotrophin (hMG), did not have an effect on testicular leukaemic infiltration. These studies demonstrate that the leukaemic infiltration of the testis is influenced by the changes in the physiological activity of the testes.

Effect of pravastatin in mildly hypercholesterolemic young men on serum matrix metalloproteinases.

Pravastatin decreases serum MMP-9 concentration in clinically healthy men. This may reflect reduction of nonsymptomatic chronic arterial inflammation.

Ovarian-adrenal cross-talk in polycystic ovary syndrome: evidence from wedge resection.

OBJECTIVE: To determine whether the ovary influences adrenal androgen secretion in women with polycystic ovary syndrome (PCOS). DESIGN: Six PCOS-affected patients with clomiphene resistance and gonadotrophin hyperresponsivity, and six controls with regular ovulatory cycles, matched for age and body mass index. METHODS: Bilateral ovarian wedge resection was performed to induce ovulation surgically for these refractory women with PCOS. The adrenal androgen secretions were evaluated in PCOS patients before and again 6 months after this surgery, and in the controls, using an ACTH stimulation test (0.25mg synthetic ACTH(1-24)). RESULTS: Biochemically, basal levels and the maximum net increases (Delta) of 17-hydroxyprogesterone (17-OHP) and androstenedione, Delta17-OHP/Delta progesterone and Delta androstenedione/Delta17-OHP ratios in response to exogenous ACTH were significantly higher in PCOS patients before operation than those of controls. This purely ovarian surgery in women with PCOS was found to significantly reduce their basal androstenedione, testosterone and LH levels, insulin/glucose ratio, and post-corticotrophic Delta17-OHP, Delta androstenedione, Delta17-OHP/Delta progesterone and Delta androstenedione/Delta17-OHP, without obvious changes in FSH, oestradiol, sex hormone-binding globulin, Delta dehydroepiandrosterone, Delta dehydroepiandrosterone sulphate, Delta aldosterone and Delta cortisol values. CONCLUSIONS: Ovarian hyperandrogenicity from polycystic ovary may contribute to the enhanced adrenal P450c17alpha activity and subsequent Delta(4) androgen reserve revealed by the pharmacological corticotrophin stimulation in our special PCOS cases.

Activation of the immune system in the testis.

The present concept for the mechanism of prolonged transplant survival in the testis suggests that lymphocyte activation is inhibited locally in this site by testicular products. As the testis produces several immunoregulatory factors, the behaviour of lymphocytes in the testis may depend on the net effect of all these factors on intralymphocytic events. In the present article, the extra- and intracellular events associated with lymphocyte activation are reviewed and the steps of lymphocyte activation which might be subject to interference by testicular factors are identified.

Testicular immunosuppressive protein.

Auto-, allo- and xenografts of various endocrine tissues survive for prolonged periods in the testicular interstitium. The reason for transplant survival outside the blood-testis barrier has been obscure. In the present paper we describe a high molecular weight (Mr = 130,000), heat- and pH-labile immunosuppressive factor with an isoelectric point of 6.3-7.3 in extracellular fluid collected from the rat testicular interstitium. The results show that the testicular immunosuppressive agent is not a steroid, but a protein. This testicular immunosuppressive protein may contribute to the immune privilege in the testicular interstitium.

Immunology of the testicular excurrent ducts.

The sperm autoantigen concentration in the epididymis equals or exceeds that in the testis. This makes the epididymis a probable site of initiation of an antisperm autoimmune response. The mechanisms regulating antisperm antibody formation in the testicular excurrent ducts and some related aspects with clinical interest are reviewed.

Autoantigenicity of the basal compartment of seminiferous tubules in the rat.

The expression of testicular autoantigens has been thought to be restricted to the luminal compartment of the seminiferous tubules. In the present study it was observed that germ cells in the basal compartment of seminiferous tubules and lamina propria of the seminiferous tubule bound antibodies in anti-testis immunosera in vivo after passive transfer of the sera by intra-peritoneal injection. The anti-testis immunosera were prepared in orchiectomised rats to avoid pre-collection adsorption of antibodies in the basal compartment of the seminiferous tubules. When testis-immune sera collected from non-orchiectomised rats or normal sera were transferred no such binding of antibodies to the basal germ cell surface or the lamina propria occurred. In Western blot analysis the anti-testis immune sera prepared in orchiectomised rats defined polypeptides from the adult rat testis with relative MWs of 19-23, 26-28, 30, 34, 38, 40-43, 45-47, 51-52, 56-57, 68, 78 and 97 kDa. The 40-43 kDa band was not detected by sera prepared in non-orchiectomised rats, suggesting that this autoantigen was expressed in the basal compartment of the seminiferous tubules. These observations suggest that the segregation of testicular autoantigens in the luminal compartment of seminiferous tubules is not complete for all of them in the rat and emphasize the role of more dynamic mechanisms in prevention of anti-germ cell autoimmune disease.

Vascular permeability to effectors of the immune system in the male rat reproductive tract at puberty.

The net movements and distribution volumes of IgG in the pubertal rat testis and epididymis were measured using physiological techniques. A new method to calculate vascular permeabilities was developed. The volume of testicular extracellular fluid accessible to IgG (Veq, microliters/g) increased between 20 and 30 days of age and remained above 20-day-old levels. Estimates of the surface areas of the exchange vessels (S, cm2) increased with age, most between 20 and 30 days of age. The speed at which equilibrium between tissue extracellular fluid and serum was reached (K, min-1) increased between 20 and 30 days and decreased between 44 and 60 days of age. The lymph flow (QL) and estimates of permeabilities (P) to IgG increased between 20 and 30 days of age and remained high. In the caput epididymidis Veq increased between 30 and 60 days and S between 44 and 60 days of age, but K increased between 30 and 44 days and decreased between 44 and 60 days of age. QL and the estimates of P increased between 20 and 44 days of age and remained at that level. In the corpus epididymidis, Veq did not change with age, but S increased between 30 and 60 days and K, QL and P between 20 and 44 days of age. In the cauda epididymidis, there were no obvious changes in Veq with age, but the estimates of S increased and K values decreased between 44 and 60 days of age. QL and P increased between 30 and 44 days and then decreased. After 30 days of age the values for Veq, K, QL and S were larger in the testis than in the cauda, whereas P at 44 days of age was not different. The number of perivascular mononuclear cell infiltrate profiles per 1000 epididymal tubule cross-sections increased with age in the caput epididymidis, but no infiltrates were observed in the corpus or cauda before 60 days of age. It is concluded that the observed increases in vascular permeability to IgG with age in the testis, caput and corpus reflect the function of the microvessels rather than an increase in the size of the vascular bed.

Expression of antigens involved in the presentation of lipid antigens and induction of clonal anergy in the female reproductive tract.

The molecular backgrounds of the anti-phospholipid syndrome and immunisation against the Rhesus proteolipid antigens are still largely unknown. In the present study, expression of (1) CD1, a major histocompatibility complex class l-like lipid antigen presenting molecule, (2) IL-10, a cytokine promoting induction of clonal anergy, and (3) CD80 and CD86, two T-cell costimulators preventing induction of clonal anergy when present, was investigated in frozen sections of cervix, corpus and the fallopian tube (FT) of 25-day-old BALB/c mice injected with FSH, progesterone or medium and of pregnant mice from each trimester (days 7, 14 and 19). CD1 was expressed by all endometrial epithelial cells. Enhanced immunostaining of the endometrial epithelial cells was observed after FSH treatment, and cervix and FT expressed generally more than the corpus of the uterus. After treatment with medroxy progesterone acetate (MPA), expression of CD1 by the endometrial epithelia was weak. During pregnancy CD1 was absent from the endometrium adjacent to the foetus, but was unaltered in the cervix and FT. IL-10 was expressed by the endometrial glands and also by the endometrial surface epithelium. MPA treatment increased the intensity of the IL-10 immunofluorescence. There were also chains of positive cells between the muscle bundles within the pregnant myometrium. CD80 and CD86 were usually absent from the female reproductive tract, but were occasionally found in the cervix during pregnancy. The present study demonstrates definite differences in the expression of both CD1 and IL-10 between the FSH and MPA treated mice, suggesting differences during the oestrous cycle. As IL-10 is expressed more in the secretory phase, it is probably involved in making the endometrium more acceptable for implantation by inducing clonal anergy. This is supported by the absence of CD80 and CD86. These results also suggest that abnormal expression of CD1d during pregnancy may predispose the mother for immunisation against lipid antigens such as membrane phospholipids and Rhesus-antigens.

Testicular immunoregulatory factors.

The present data indicate that immune cells are regulated locally in the testis by Leydig cells, Sertoli cells and resident testicular macrophages. The effects of these cells are mediated by several peptide factors, including protectin, a group of high molecular weight testicular immunosuppressive factors, and testicular interleukin-1 alpha-like factor. The testicular interleukin-1 alpha-like factor is produced by Sertoli cells and is under hypophyseal control. Its synthesis starts at puberty concomitantly with the onset of spermatogenesis and it may act as a spermatogonial growth factor. Protectin, which is under hypophyseal control, may be involved in the mechanism of prolonged transplant survival in the testicular interstitial tissue. Its levels increase at puberty. Both the testicular interleukin-1 alpha-like activity and protectin may be important in testicular pathophysiology.

Testicular immunosuppressive activity in experimental hypogonadism and cryptorchidism.

The levels of protectin activity were studied in scrotal and abdominal testes and in testes of hypophysectomized and hormone-treated rats to determine whether the prolonged survival of transplants in the abdominal testis was associated with higher protectin activity and whether the protectin activity was dependent on testosterone production. The levels of the testicular high-molecular weight immunosuppressive activity per gram of tissue or per protein concentration rose progressively in the abdominal testis until 7 days after the rats had been made unilaterally cryptorchid. No such increase was observed in the contralateral scrotal testis. In hypophysectomized rats the levels of the testicular immunosuppressive activity were higher than in normal controls. Increase in the serum testosterone concentrations in the hypophysectomized rats obtained by daily treatment with human chorionic gonadotrophin or pregnant mare serum gonadotrophin was associated with significantly lower levels of the testicular immunosuppressive activity as compared to untreated hypophysectomized animals. In the normal rats estradiol treatment increased the levels of testicular immunosuppressive activity as serum testosterone concentrations decreased. In gel filtration there were clear peaks of immunosuppressive activity eluting at 25, 32 and 44 min, corresponding to Mr of greater than 400,000, 200,000 and 25,000, respectively. These results suggest that the levels of testicular immunosuppressive activity increase when testosterone production decreases and support the earlier suggestion that transplant survival in the rodent testis is not dependent on the maintenance of normal testosterone levels in the recipient testis.

Isolation and partial characterization of an interleukin-1-like factor from rat testis interstitial fluid.

Testicular interstitial fluid (ISF) was collected by in vivo perfusion of rat testes and analyzed for the presence of interleukin-1 (IL-1) activity by utilizing a murine thymocyte proliferation assay. IS obtained from nine rats were all positive with dose-response curves of IL-1 activity similar to those produced by rat testicular aqueous extracts, rat macrophage IL-1 and human recombinant IL-1 alpha. Chromato-focusing of pooled ISF revealed a single peak of IL-1 activity with an estimated isoelectric point of 6.1-6.3. HPLC size exclusion chromatography demonstrated two active peaks with apparent molecular ratios Mr of 15,000-18,000 and 5000-7000, respectively. The molecular properties of the 15,000-18,000 Mr component are very similar to those of an IL-1-like factor previously isolated from seminiferous tubules. Our results indicate that the testicular IL-1-like factor is secreted by the seminiferous tubules into the interstitial tissue. Its function in the testicular interstitium is unknown but it might be relevant for the tendency to testicular relapse of childhood lymphocytic leukemia.

Ontogeny of immunosuppressive activity, MHC antigens and leukocytes in the rat testis.

The levels of immunosuppressive activity and the presence of MHC antigens and leukocytes were studied in the immature and the sexually mature rat testis. The immunosuppressive activities were measured from high-molecular weight (greater than 5 kDa) fractions of testis extracts using the protectin bioassay. The presence of MHC antigens and leucocytes was studied using the indirect immunoperoxidase method. In the immature rats, clusters of class I MHC antigen positive cells and a few cells expressing class II MHC antigen were present in the testicular interstitium. In the sexually mature rats, all the cells were MHC I+, and MHC II+ cells were numerous in the testicular interstitium. The seminiferous epithelium was MHC-negative in both the immature and the sexually mature testis. W3/25+ leukocytes were present in the interstitium and the tubular wall in both the immature and the sexually mature rat testis, but not in the seminiferous epithelium at any age. At 20-30 days of age, the testicular extracts were neutral or slightly stimulated 3H-TdR incorporation into peripheral blood lymphocytes, but at 44-60 days of age they inhibited lymphocyte proliferation significantly. In gel filtration, a peak of immunosuppressive activity was observed at approximately 400 kDa (protectin A) in both 20- and 60-days-old rat testes. A smaller peak was present at approximately 200 kDa in both age groups. This study shows that the testicular immunoregulatory microenvironment is different in the immature and the sexually mature rats. This may be important in such age-dependent human diseases as mumps orchitis and the testicular relapses of acute lymphoblastic leukemia.

Di-n-pentyl phthalate-induced inflammatory changes in the rat testis are accompanied by local production of a novel lymphocyte activating factor.

Phthalate esters (PAE) are plasticizers in polyvinyl chloride plastics used, for example, in package material for medical solutions. PAE exposure is associated with testicular damage that primarily affects Sertoli cells, and is concomitant with leukocyte infiltration into the testicular interstitium. We have demonstrated that the rat testis constitutively produces a lymphocyte activating factor (LAF) resembling interleukin-1 alpha, and originating from Sertoli cells. The testicular interleukin-1-like factor (tIL-1) has a relative molecular mass (Mr) of 17,000 (17 k) and an isoelectric point (pI) of 5.7. In the present study we have measured testicular LAF activity after exposure to di-n-pentyl phthalate (DPP) in 40-day-old rats. We found a large increase in LAF bioactivity which was evident already 3 h after a single oral dose of DPP. The increase was maximal 9-12 h after exposure, and had decreased toward the control level at 24 h. The increased activity was found to be at least partly due to the induction of a novel LAF with Mr 38,000 and pI 4.5. Morphological examination confirmed earlier results with an interstitial leukocyte infiltration 6 h after DPP exposure. The identity of the novel LAF and its functional relation to testicular inflammation remain to be established.

CD28-CD80/CD86 interactions in testicular immunoregulation.

The expression of two accessory molecules on antigen-presenting cells (APC), the CD80/ B7-1 and CD86/B7-2 antigens, was studied in the testis of normal and non-obese diabetic (NOD) mice. In addition, the effect of CD28 stimulation on suppression of lymphocytes by testicular products was investigated. The testes of 4-week old NOD mice or normal BALB/c mice and the testis of 17-21-week old BALB/c mice contained no CD80 or CD86 expressing cells. In contrast, CD80+ and CD86+ cells were present in the testis of 14-22-week old NOD mice. The CD80+ cells and most of the CD86+ cells were CD11b/CD18 negative. There were some CD11b/CD18+ cells that expressed CD86 weakly. The CD80+ and CD86+ cells were often located adjacent to the vessel walls where a leukocyte not expressing CD80 or CD86 had attached to the endothelium. Some CD80+ and CD86+ cells were present among the interstitial cells. The CD80 and CD86 antigens could not be observed in the same cells as judged from stainings in parallel sections. Stimulation of ConA- or anti-CD3 epsilon-primed peripheral blood or spleen lymphocytes with anti-CD28 was able significantly to antagonize the growth-inhibitory effect of the M(r) > 5 K fraction of testis extracts, but could not abolish it with increasing concentrations of testis extract. The results suggest that T lymphocytes can not be activated locally in the testis of BALB/c and young NOD mice because of the absence of the necessary CD28 ligands, CD80 and CD86, from the APCs and because of the suppression of T lymphocytes by the testicular products. In the testis of older diabetic NOD mice lymphocyte activation may occur because the testes of these mice contain CD80+; CD11b/CD18-, CD86+; CD11b/CD18+ and CD86+; CD11b/CD18- cells and therefore, CD28 co-stimulation, which can antagonize the suppressive effect of testis extract, may occur. The possibilities for clonal anergy in testicular immunoregulation are discussed.

Testis physiology relevant to immunoregulation.

Intra-testicular transplants are placed in rodents into the large lymphatic sinusoids of the interstitial tissue of the testis. These sinusoids are filled with a protein-rich extracellular fluid that supplies all the requirements of the grafts until vascularization takes place. The testicular microvascular endothelium regulates transport of T lymphocytes and immunoglobulin G to the testis and may thus contribute to regulation of the immune system in this organ. Differences in the organization of the lymphatic drainage exist between species, but in every studied species lymphatic drainage from the testis leads to lymph nodes.

Regulation of the testis.

The testicular cells are regulated by factors produced locally in the testis. These factors include peptide growth factors, pro-opiomelanocortin derivatives, neuropeptides and steroids. Several agents able to affect steroido- and spermatogenesis can also affect leukocytes and many of the testis-regulating factors are produced by immune cells, suggesting that testicular cells and leukocytes may interact. In the present article, the effects of various testicular cell and leukocyte produced factors on steroido- and spermatogenesis are reviewed. The possibility that leukocytes may produce substances able to affect the testicular functions suggests that inhibition of immune system activation in the testis may be important also for reasons other than protection of autoantigenic germ cells from an autoimmune attack.

Regulation of the transport of immunoglobulin G into the male rat reproductive tract.

The net movements of IgG in the rat testis and epididymis were studied using physiological techniques in conditions of anticipated subnormal organ concentrations of testosterone (T). The volume of extracellular fluid accessible to IgG per gram whole testis (Veq, microliter/g) did not change but that per gram interstitial tissue decreased in intact rats treated with T to suppress gonadotrophin secretion. Estimates of surface areas of the exchange vessels (S, cm2/g whole tissue) did not change in any of the organs studied. The speed at which equilibrium between tissue extracellular fluid and serum was reached (magnitude of K, min-1), lymph flow (QL, microliter/g/min) and estimates of microvascular permeability (P, nm/min) to IgG decreased in the testis of T-treated intact rats. In these animals, magnitude of K and P were higher than normal in the cauda. After castration, magnitude of K and P decreased to near zero in the caput and corpus regions. The changes in magnitude of K and P due to castration could partly be prevented by T supplementation in the caput, but not in the corpus, where Veq was supra-normal after T treatment. This difference in endothelial response between caput and corpus indicates differences in control of endothelium and epithelium along the length of the epididymal duct. In the testis of efferent duct-ligated (EDL) rats, P was lower than that in the intact rats, whereas in the corpus epididymidis of EDL rats, Veq and P were higher than normal. The supra-normal Veq values in the corpus epididymidis of EDL and castrated T-treated rats suggest that the epithelial barrier leaks in the corpus when luminal testosterone is low. It is concluded that during testosterone administration to normal rats, the testicular and epididymal epithelial barrier remains intact, that trans-endothelial transport of IgG and extracellular fluid volume decrease in the testicular interstitium and that in conditions of low serum T, the endothelium of microvessels in the caput and corpus epididymidis forms a barrier to IgG.