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Summary Recently, IL-18 was identified in human testes. Moreover, an inverse correlation was found between the levels of IL-18 and the number and motility of spermatozoa. We examined the presence of IL-18 protein in normal and impaired spermatogenesis. Testicular tissue specimens were taken from 25 nonobstructive azoospermic patients undergoing testicular sperm extraction and from autopsies of three healthy controls. The presence of IL-18 in human testicular cells was examined by immunohistochemical staining of paraffin-embedded sections, using a specific antibody for human IL-18. In testicular tissue of healthy controls as well as in study cases, presence of IL-18 was identified in somatic, mitotic, meiotic and post-meiotic cells in correlation with their presence. In all patients, Leydig cells were less intensively stained. Mitotic cells were immunostained in the control group and less intensively in hypospermatogenesis and maturation arrest subgroups. Primary spermatocytes were in general most efficiently stained. The expression of IL-18 mRNA (as examined by real-time PCR analysis) showed significantly lower expression in testicular tissues with impaired spermatogenesis when compared to normal tissues. We report the first study demonstrating the presence of IL-18 in human testicular tissue at the protein level. The presence of this cytokine in somatic as well as in different types of germ cells may suggest its involvement in the regulation of the spermatogenic process and steroidogenesis under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

The present study focuses on evaluating the potential of flattened AgClBr fibre-optic evanescent wave spectroscopy (FTIR-FEWS) technique for detection and identification of cancer cells in vitro using cell culture as a model system. The FTIR-FEWS results are compared to those from FTIR-microspectroscopy (FTIR-MSP) method extensively used to identify spectral properties of intact cells. Ten different samples of control and malignant cells were measured in parallel by the above two methods. Our results show a significant similarity between the results obtained by the two methodologies. The absorbance level of Amide I/Amide II, phosphates and carbohydrates were significantly altered in malignant compared to the normal cells using both systems. Thus, common biomarkers such as Amide I/Amide II, phosphate and carbohydrate levels can be derived to discern between normal and cancer cells. However, marked differences are also noted between the two methodologies in the protein bands due to CH3 bending vibration (1480–1350 cm−1). The spectral differences may be attributed to the variation in the penetration depth of the two methodologies. The use of flattened fibre rather than the standard cylindrical fibre has several practical advantages and is considered as an important step towards in vivo measurements in real time, such as that of skin nevi and melanoma using special designs of fibre-optic–based sensors. [ABSTRACT FROM AUTHOR]

Problem: This study examined the effect of intraperitoneal administration of lipopolysaccharide (LPS) to adult male mouse on the expression levels of interleukin-18 (IL-18), IL-18 receptor (IL-18R) and the IL-1beta converting enzyme (ICE) (IL-18 family) in their testes and spleen (control)., Method of Study: Adult mice were injected (intraperitoneally; i.p.) with saline (control) or LPS (2, 20, 100 microg/mL; 100 microL/mouse). After 3 and 24 hr, testes and spleen were collected. Testicular tissue was examined for IL-18 (by ELISA, real time PCR, and western blot analysis), IL-18R and ICE (western blot and real time PCR analysis) and spleen tissue was examined for the IL-18 family by real time PCR analysis. Student's t-test was used for statistical analysis., Results: Homogenates of mouse testes contain and express basal levels of IL-18, ICE and IL-18 R. The expression levels of IL-18, ICE and IL-18R were significantly increased 3 and 24 hr after intraperitoneal injection of LPS to mature mouse, as examined by ELISA, western blot and real time PCR analysis. However, the expression levels of IL-18, ICE and IL-18Ralpha in the spleen increased significantly only after 24 hr of LPS stimulation, as examined by real time PCR., Conclusion: Our results demonstrate that LPS increases the expression levels of the IL-18 family in mouse testis and spleen, but the time of expression differs between the two organs. The presence of IL-18 in the testes might be involved in the regulation of physiological and infection/inflammatory processes, and may be part of the autocrine/paracrine factors that control spermatogenesis. Further studies should be performed to confirm this possibility.

Problem: This study examined the effect of lipopolysaccharide (LPS) on the capacity of Leydig cells to produce and express interleukin-18 (IL-18), IL-18 receptor (IL-18R) and the IL-1beta-converting enzyme (ICE) (IL-18 family), under in vitro conditions., Method of Study: Primary Leydig cells (LCs) were isolated from murine testis by the Percoll technique, and cultured both in the presence and absence of LPS (0.1, 1, 5 microg/mL) for 3 and 24 hr. LCs were examined for their capacity to produce and express IL-18 family molecules by using immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA), Western blot and real-time polymerase chain reaction (PCR) analysis., Results: Leydig cells were shown to constitutively express IL-18, as examined by IHC, ELISA, Western blot and real-time PCR analysis. Addition of LPS to LC cultures was shown to significantly increase the basal levels of IL-18, in a dose- and time course-dependent manner, as examined by ELISA, Western blot and real-time PCR analysis. In addition, LPS increased LC cultures to express ICE and IL-18 R, as examined by real-time PCR analysis., Conclusion: Our results demonstrate that LPS increased the capacity of murine LCs to produce the IL-18 family molecules. IL-18, in the testis, might be involved in the regulation of physiological and infection/inflammatory processes, and thus, could be a component of the autocrine/paracrine factor net that controls steroidogenesis and male fertility; further studies are needed to confirm this possibility.

Hypoxia (H) and hypoxia-ischemia (HI) are major causes of foetal brain damage with long-lasting behavioral implications. The effect of hypoxia has been widely studied in human and a variety of animal models. In the present review, we summarize the latest studies testing the behavioral outcomes following prenatal hypoxia/hypoxia-ischemia in rodent models. Delayed development of sensory and motor reflexes during the first postnatal month of rodent life was observed by various groups. Impairment of motor function, learning and memory was evident in the adult animals. Activation of the signaling leading to cell death was detected as early as three hours following H/HI. An increase in the counts of apoptotic cells appeared approximately three days after the insult and peaked about seven days later. Around 14-20 days following the H/HI, the amount of cell death observed in the tissue returned to its basal levels and cell loss was apparent in the brain tissue. The study of the molecular mechanism leading to brain damage in animal models following prenatal hypoxia adds valuable insight to our knowledge of the central events that account for the morphological and functional outcomes. This understanding provides the starting point for the development and improvement of efficient treatment and intervention strategies.

Problem: Interleukin-1 family is present in the testicular homogenates and its cellular compartments. It has been suggested that IL-1 is involved in physiological and pathological functions of the testicular tissues. In the present study we examined the effect of acute mostly localized inflammation, using turpentine, on the expression levels of testicular IL-1 system., Methods of Study: Mice were subcutaneously injected with steam-distilled turpentine or saline (control). Three hours to 10 days following the injection, mice were killed and testis and spleen were homogenized and examined for interleukin (IL)-1alpha, IL-1beta, and IL-1 receptor antagonist (IL-1ra) levels by enzyme-linked immunosorbent assay and polymerase chain reaction., Results: Subcutaneous injection of turpentine induced mice systemic inflammation, as indicated by significant increase in serum IL-1beta levels, and IL-1alpha, IL-1beta and IL-1ra in spleen homogenates. The levels of IL-1alpha, IL-1beta and IL-1ra were significantly induced in testicular homogenates of adult mice following subcutaneous injection of turpentine. The significant induction of testicular IL-1alpha was detected after 3-24 hr of turpentine injection and decreased later (after 3-10 days) to levels similar to the control. However, significant induction of testicular IL-1beta was detected only after 3-10 days of turpentine injection, and for testicular IL-1ra levels was detected after 3 hr to 6 days of turpentine injection, and after 10 days the levels were similar to the control. These results were also confirmed by mRNA expression of these factors., Conclusion: Our results demonstrate for the first time the distant effect of acute localized inflammation on testicular IL-1 levels. Thus, transient inflammatory response to infectious/inflammatory agents at non-testicular sites that elicit systemic IL-1 response should be considered during clinical treatment as a possible factor of male infertility.

Problems: To determine if magnesium sulfate (MgSO4) attenuates the vasoconstrictor effect of angiotensin II (Ag II), endothelin-1 (ET-1) and thromboxane mimetic (TX) in the human fetal placental vasculature and if interleukin-1 beta (IL-1beta) is involved in this process., Study Design: Isolated placental cotyledons (n = 18) were dually perfused with fetal perfusion pressure used as an index of vascular response. The vasoconstriction effect of bolus injection of various concentrations of Ag II (10(-1)) 10(-6) M) or ET-1 (10-(10)-10(-4) M) or TX (10(-10)) 10(-5) M) was established in the absence or presence of MgSO4 (7 mg% constant infusion during 10 hr). Statistical significance was determined by paired t-test and ANOVA., Results: MgSO4 significantly attenuates the vasoconstrictor effect of Ag II in the fetal placental vasculature in the human placenta (P = 0.02). Moreover, significant attenuation of vasoconstrictor response to ET-1(10(-10))10(-5) M) was observed in the presence of MgSO4 (P = 0.02). However, no attenuation of the vasoconstrictor response to TX was noted in the presence of MgSO4 (P > 0.5). Ag II and TX were shown to induce IL-1beta secretion by placental tissue. This effect was completely reduced by perfusion of MgSO4 (7 mg%; constant infusion)., Conclusions: MgSO4 significantly attenuates the vasoconstrictor effect of Ag II and ET-1 in the fetal-placental vasculature in the human placenta, but not that of TX. Inhibition of local production of IL-1beta could be one of the mechanisms used by MgSO4 to reduce the vasoconstrictory effect of Ag II and TX in human placental cotyledone.

Problem: Uterine artery was shown to be unique in its capacity to change in size and function during pregnancy. As interleukin-1 (IL-1) was shown to be involved in reproduction processes, the aim of this study was to determine the levels of IL-1 activity of the uterine artery tissue in pregnant rat., Method of Study: Nine virgins and nine midpregnant rats were selected. Both uterine arteries were obtained, together with reference tissues from aorta and uterus. The levels of IL-I were examined in the above tissues after culturing with media alone (control; CT), and media that contained stimulants like tumor necrosis factor-alpha (TNF-a) or lipopolysaccharide (LPS). IL-1-like activity was evaluated by its capacity to promote the culture growth of 1A-5 and cytotoxic T lymphocyte derived (CTLD) cell lines. This activity was expressed as optical density (OD)/mg protein of the examined organ., Results: Uterine artery tissue, of pregnant rats, cultured in medium alone produced significantly higher levels of IL-1 than uterine artery of virgin animals under the same conditions (16.2 S.E. 1.3 versus 0.6 S.E. 0.05 OD/mg protein, respectively; P < 0.02). Stimulation of uterine artery in vitro by LPS and TNF increased their capacity to secrete IL-1. In comparison with uterine artery, aorta produced higher levels of IL-1 in virgin rats compared with pregnant rats (13.6 S.E. 1.2 versus 1.6 S.E. 0.1; P < 0.02). Stimulation of aorta tissues (from both virgin and pregnant rats) with LPS, in vitro, significantly decreased their capacity to secrete IL-1 (P < 0.04). Stimulation of aorta tissues from virgin rats with TNF-alpha, in vitro, did not change their capacity to secrete IL-1 activity. However, stimulation of aorta tissues from pregnant rats with TNF-alpha decreased the secretion of bioactive IL-1. The levels of IL-1 produced by uterine tissues from virgin and pregnant rats were similar, and stimulation with either LPS or TNF-alpha significantly decreased their capacity to secrete IL-1 (P < 0.04)., Conclusions: The high level of IL-1 activity detected during pregnancy in the uterine artery may suggest its unique involvement in the changes occurring throughout pregnancy in those blood vessels.

In the present study we examined the capacity of interleukin-1 (IL-1) alpha, beta, interleukin-1 receptor antagonist (IL-1ra) and follicle stimulating hormone (FSH) to induce transferrin secretion by Sertoli cells under in vitro conditions. Primary Sertoli cell (SC) cultures from immature mice secreted constitutively transferrin. Stimulation of these cultures with IL-1alpha, IL-1beta significantly increas\d their capacity to secrete transferrin. Addition of IL-1ra to unstimulated SC cultures did not affect their capacity to secrete transferrin. Stimulation of SC cultures with a combination of both IL-1alpha and FSH or IL-1beta and FSH showed additive effect between IL-1 and FSH in their capacity to induce transferrin secretion by these cells. However, stimulation of Sertoli cells with a combination of both IL-1ra and FSH did not affect their capacity to secrete transferrin compared with FSH-stimulated cultures. Our results may suggest the involvement of testicular paracrine/autocrine factors (IL-1) and endocrine (FSH) factors in the regulation of transferrin secretion by SC. This capacity seems to be differently regulated by these factors. Thus, IL-1alpha and beta may directly affect physiological functions of the testis; which may suggest their involvement in the regulation of spermatogenesis and spermiogenesis processes and male fertility.

Problem: To examine the capacity of sperm cells from fertile and infertile men to secrete interleukin (IL)-6, and the involvement of serum factors and lipopolysaccharide (LPS) in the regulation of IL-6 and IL-1 production by sperm cells., Methods: Swim-up sperm cells from fertile (donors) and oligoteratoasthenospermic (OTA)-infertile men were incubated with or without 5% fetal calf serum (FCS) and LPS (10 microg/mL) for 2-24 hr. After incubation, IL-6 and IL-1 bioactivities were measured in supernatants and lysates by specific bioassays (B9 cell proliferation assay and 1A-5 system, respectively)., Results: IL-6- and IL-1-like activities were observed to be produced by swim-up sperm cells from both study groups. Stimulation of swim-up sperm cells with either LPS or FCS or both together did not affect their capacity to produce IL-1. However, LPS, but not serum increased the secretion levels of IL-6 by swim-up sperm cells., Conclusions: Swim-up sperm cells from both study groups constitutively produce IL-6 and IL-1, and serum components did not affect this capacity. However, LPS was shown to increase the capacity of swim-up sperm cells of both study groups to secrete IL-6, but not IL-1. Cytokines may be involved in the physiology and pathophysiology of sperm functions and, thus, may affect male fertility.

Problem: To assess the effect of seminal plasma (SP) of fertile and infertile men on leukocyte mitogenic response, and the capability of sperm cells to produce IL-1., Methods: This study included four groups: fertile men (donors, normal), infertile men with azoospermia (azoo), oligo-terato-asthenozoospermia (OTA), and OTA with genital infection (OTA-inf). Mouse spleen cell proliferation in response to lipopolysaccharide (LPS) or Concanavalin-A (Con-A) was examined in the presence of SP from the above four groups. Supernatants (sup) and lysates (lys) of sperm cells from fertile and oligoteratoasthenospermic (OTA) men were evaluated for IL-1 bioactivity by specific bioassay., Results: Seminal plasma (SP) of the four groups were shown to inhibit the mitogenic response of mouse spleen cells to LPS and Con-A. SP of fertile men was significantly more inhibitory than SP from infertile men. Sperm cells from fertile and OTA infertile men constitutively produced IL-1. Sperm cells of both groups produced similar levels of IL-1 as examined in the supernatants and lysates., Conclusions: Seminal plasma of fertile men had more inhibitory mitogenic activity than that of OTA. Sperm cells constitutively produce IL-1. It is possible that the factors involved in this inhibition are not only anti-proliferative immune factors. Cytokines and inhibitory factors of mitogenesis in the seminal plasma may be involved in the physiology and pathophysiology of sperm functions and thus affect male fertility.