Biomarkers of cell damage, neutrophil and macrophage activation associated with in-hospital mortality in geriatric COVID-19 patients.

BACKGROUND: The risk for symptomatic COVID-19 requiring hospitalization is higher in the older population. The course of the disease in hospitalised older patients may show significant variation, from mild to severe illness, ultimately leading to death in the most critical cases. The analysis of circulating biomolecules involved in mechanisms of inflammation, cell damage and innate immunity could lead to identify new biomarkers of COVID-19 severity, aimed to improve the clinical management of subjects at higher risk of severe outcomes. In a cohort of COVID-19 geriatric patients (n= 156) who required hospitalization we analysed, on-admission, a series of circulating biomarkers related to neutrophil activation (neutrophil elastase, LL-37), macrophage activation (sCD163) and cell damage (nuclear cfDNA, mithocondrial cfDNA and nuclear cfDNA integrity). The above reported biomarkers were tested for their association with in-hospital mortality and with clinical, inflammatory and routine hematological parameters. Aim of the study was to unravel prognostic parameters for risk stratification of COVID-19 patients. RESULTS: Lower n-cfDNA integrity, higher neutrophil elastase and higher sCD163 levels were significantly associated with an increased risk of in-hospital decease. Median (IQR) values observed in discharged vs. deceased patients were: 0.50 (0.30-0.72) vs. 0.33 (0.22-0.62) for n-cfDNA integrity; 94.0 (47.7-154.0) ng/ml vs. 115.7 (84.2-212.7) ng/ml for neutrophil elastase; 614.0 (370.0-821.0) ng/ml vs. 787.0 (560.0-1304.0) ng/ml for sCD163. The analysis of survival curves in patients stratified for tertiles of each biomarker showed that patients with n-cfDNA integrity < 0.32 or sCD163 in the range 492-811 ng/ml had higher risk of in-hospital decease than, respectively, patients with higher n-cfDNA integrity or lower sCD163. These associations were further confirmed in multivariate models adjusted for age, sex and outcome-related clinical variables. In these models also high levels of neutrophil elastase (>150 ng/ml) appeared to be independent predictor of in-hospital death. An additional analysis of neutrophil elastase in patients stratified for n-cfDNA integrity levels was conducted to better describe the association of the studied parameters with the outcome. CONCLUSIONS: On the whole, biomarkers of cell-free DNA integrity, neutrophil and macrophage activation might provide a valuable contribution to identify geriatric patients with high risk of COVID-19 in-hospital mortality.

Main biomarkers associated with age-related plasma zinc decrease and copper/zinc ratio in healthy elderly from ZincAge study.

PURPOSE: Zinc (Zn) plays an essential role in many biological processes including immune response. Impaired Zn status promotes immune dysfunction, and it has been associated with enhanced chronic inflammation during aging. It has been suggested that the measurement of circulating Zn by itself could not reflect the real Zn status of an individual. It is therefore necessary to identify other determinants associated with plasma Zn to better understanding how physiopathological conditions during aging may affect the concentration of this metal. METHODS: We have investigated the association between Zn levels and some biomarkers in 1090 healthy elderly from five European countries to increase the accuracy in the assessment of the Zn status. Stepwise multivariate linear regression models were used to analyze the influence of factors such as age, dietary intake, inflammatory mediators, laboratory parameters and polymorphisms previously associated with Zn homeostasis. RESULTS: Plasma Zn decrement was most strongly predicted by age, while positive correlations were found with albumin, RANTES and Zn intake after adjustment for multiple confounders. HSP70 +1267 AA genotype was an independent factor associated with Zn plasma concentrations. Cu/Zn ratio was positively associated with markers of systemic inflammation and age and negatively associated with albumin serum levels. CONCLUSIONS: Our findings show the most important independent determinants of plasma Zn concentration and Cu/Zn ratio variability in elderly population and suggest that the decline with age of Zn circulating levels is more dependent on physiopathological changes occurring with aging rather than to its nutritional intake.

EFFECT OF PLATELET RICH PLASMA CONCENTRATION ON SKELETAL MUSCLE REGENERATION: AN EXPERIMENTAL STUDY.

Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for up to 55% of all sports injuries. The phases of the healing processes after direct or indirect muscle injury are complex but clearly defined and include well-coordinated steps: degeneration, inflammation, regeneration, and fibrosis. Despite this frequent occurrence and the presence of a body of data on the pathophysiology of muscle injuries, none of the current treatment strategies have shown to be really effective in strictly controlled trials. Platelet-rich plasma (PRP) is a promising alternative approach based on the ability of autologous growth factors (GFs) to accelerate tissue healing, improve muscular regeneration, increase neovascularization and reduce fibrosis. The present study is focused on the use of different concentrations of PRP as a source of GFs. Unilateral muscle lesions were created on the longissimus dorsi muscle of Wistar rats. Twenty-four h after surgical trauma, the lesion was filled with an intramuscular injection of PRP at 2 different concentrations. A group of rats were left untreated (controls). Animals were sacrificed at 3, 15 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The PRP-treated muscles showed better muscle regeneration, more neovascularization and a slight reduction of fibrosis compared with the control muscles in a dose dependent manner. However, further studies also assessing pain and functional recovery are scheduled.

In vitro activity and in vivo efficacy of tigecycline alone and in combination with daptomycin and rifampin against Gram-positive cocci isolated from surgical wound infection.

The aim of this work was to determine the in vitro activity of tigecycline and its bactericidal effect for a large number of Gram-positive cocci, as well as to investigate its in vitro interaction with six clinically used antibiotics. In vivo, a wound model was established through the panniculus carnosus of BALB/c mice, and then inoculated with 5 × 10(7) colony-forming units (CFU) of Staphylococcus aureus or Enterococcus faecalis. For each bacterial strain, the study included an infected or non-infected group that did not receive any treatment, three groups singly treated with tigecycline, rifampin, and daptomycin, and two groups that received tigecycline treatment plus rifampin or daptomycin. In the in vitro studies, tigecycline, daptomycin, and teicoplanin were active against all of the 48 Gram-positive isolates. The combination of tigecycline with rifampicin and daptomycin was synergistic against S. aureus and Enterococcus spp. In the in vivo studies, all groups treated with single drugs showed statistically significant results compared to the control group. The two groups treated with a combination of drugs showed the highest antimicrobial efficacy. In conclusion, our results suggested a strong activity of tigecycline alone and in combination with other antimicrobial agents against multi-resistant Gram-positive organisms isolated from wound infections.

Effectiveness of antimicrobial photodynamic therapy with a single treatment of RLP068/Cl in an experimental model of Staphylococcus aureus wound infection.

BACKGROUND: Chronic leg ulceration is a common health problem. It is well known that a clinically relevant bacterial load in chronic cutaneous wounds interferes significantly with the normal process of healing. Staphylococcus aureus is the most important representative of the staphylococcal group which causes clinically relevant infections within immunocompetent patients. OBJECTIVES: To investigate the efficacy of a single treatment of antimicrobial photodynamic therapy (APDT) with RLP068/Cl in a mouse model of a surgical wound infection induced with a methicillin-resistant strain of S. aureus (MRSA). METHODS: Wounds, established through the panniculus carnosus of BALB/c and CD1 mice, were inoculated with 5 x 10(7) c.f.u. of MRSA. Mice were randomized into four groups respectively receiving no treatment, APDT with placebo, APDT with a new phthalocyanine derivative (RLP068/Cl) and intraperitoneal teicoplanin. RESULTS: On day 2 from infection, a strong reduction of bacterial counts (≈ 3 logs) was observed in mice treated with RLP068/Cl in comparison with infected untreated mice. On day 9 from infection, a comparable and significant (≈ 2 logs) reduction of bacterial counts was found in mice treated with RLP068/Cl or with teicoplanin. At this time, histological examinations revealed that wounds treated with RLP068/Cl showed a complete re-epithelialization with a continuous epithelial lining. CONCLUSIONS: The results of the in vivo study demonstrated that APDT with RLP068/Cl may be useful in the management of chronic infected wounds, accelerating the repair process through a significant bacterial inhibition.

Persistent ex vivo low number and functional in vitro recovery of circulating gammadelta T cells after removal of a cutaneous primary melanoma.

We recently described gammadelta T cells alterations in patients with a cutaneous primary melanoma. To evaluate whether gammadelta T cells alterations persisted after melanoma removal, we performed a follow-up study comparing the number and function of gammadelta T lymphocytes from 19 subjects, 4 years after the removal of a cutaneous primary melanoma, with the data obtained in the same subjects before the surgical intervention and with control donors. The number of circulating gammadelta(+) T cells after melanoma removal was not recovered to the levels found in controls. gammadelta(+) T cells producing TNF-alpha or IFN-gamma were increased after melanoma removal in comparison with the same subjects before surgical intervention or with control donors. After in vitro culture, both the percentage and the expansion of gammadelta T cells were recovered to the values found in controls. In conclusion, the functional capacity of gammadelta T cells was in vitro recovered after melanoma removal, whereas their ex vivo number remained at lower levels than control donors.

Acetylcholinesterase inhibitors in Alzheimer's disease influence Zinc and Copper homeostasis.

BACKGROUND: Alzheimer's disease (AD) is the most common age-related neurodegenerative disease. An altered homeostasis of Zinc (Zn) and Copper (Cu), as well as a dysregulated expression of Zn-regulatory proteins have been previously described in AD. Acetylcholinesterase inhibitors (AChEI) are commonly used as AD treatment to improve cognitive function, but their effect on Zn homeostasis is still unexplored. OBJECTIVES: The aims of this study were to define the metal dyshomeostasis in AD patients, to investigate AChEI influence on Zn homeostasis and inflammation, and to analyze the relationship between cognitive impairment at two-year follow-up and metal concentrations, considering AChEI use. METHODS AND RESULTS: 84 Healthy Elderly (HE) and 95 AD patients were enrolled (62 AchEI user and 33 AchEI naïve). HE showed similar plasma Zn and Cu concentrations and Cu/Zn ratio in comparison to AChEI users, but significantly higher Zn level, as well as lower Cu amount and Cu/Zn ratio than AChEI naïve patients. Moreover, AChEI users had increased Zn plasma level, reduced Cu amount, Cu/Zn ratio, and IL1ß concentration and lower Zip2 lymphocytic expression vs. naïve patients. A multiple linear regression analysis showed that the MMSE score decline after two-year follow-up was reduced by AChEI therapy and was positively associated with plasma Zn decrease over time. CONCLUSION: Our data revealed that AChEI use may affect peripheral Zn and Cu homeostasis in AD patients, decrease Cu/Zn ratio demonstrating a general reduction of inflammatory status in patients under AChEI treatment. Finally, AChEI influence on circulating Zn could be implicated in the drug-related slowdown of cognitive decline.

ZnT8 Arg325Trp polymorphism influences zinc transporter expression and cytokine production in PBMCs from patients with diabetes.

AIMS: ZnT8 Arg325Trp polymorphism has been associated with type 2 diabetes (T2DM) susceptibility. The Arg-325 risk variant shows accelerated zinc (Zn) transport kinetic and reduced glucose-stimulated insulin secretion in pancreatic cells. However, it remains unexplored the role of Znt8 polymorphism in the regulation of Zn homeostasis and inflammatory response in peripheral blood mononuclear cells (PBMCs) from T2DM patients. METHODS AND RESULTS: A total of 556 healthy controls and 413 T2DM patients were genotyped for ZnT8 Arg325Trp polymorphism confirming the association of Arg-325 variant with an increased T2DM risk (OR = 1.35 95% C.I: 1.10-1.66; p = 0.0044). Moreover, PBMCs from Arg/Arg T2DM subjects showed increased intracellular free Zn, higher gene expression of Metallothioneins, Znt1, Znt8, Zip2 genes, and reduced Znt4 and Znt7. Higher release of IL-1α, IL-1ß, IFN-γ, IL-12p70 and TNF-α and a reduced IL-10 secretion after lipopolysaccharide (LPS) stimulation were observed in PBMCs from Arg/Arg T2DM carriers as compared to subjects with the Trp variant. CONCLUSIONS: Our data provide evidence of a substantial different Zn homeostasis regulation between Znt8 Arg-325 and Trp-325 carriers in PBMCs from T2DM patients. Moreover, Znt8 Arg-325 risk variant shows an enhanced inflammatory response upon LPS stimulation that might aggravate insulin resistance and the progression of diabetes cardiovascular complications.

Combination of Coenzyme Q10 Intake and Moderate Physical Activity Counteracts Mitochondrial Dysfunctions in a SAMP8 Mouse Model.

Aging skeletal muscles are characterized by a progressive decline in muscle mass and muscular strength. Such muscular dysfunctions are usually associated with structural and functional alterations of skeletal muscle mitochondria. The senescence-accelerated mouse-prone 8 (SAMP8) model, characterized by premature aging and high degree of oxidative stress, was used to investigate whether a combined intervention with mild physical exercise and ubiquinol supplementation was able to improve mitochondrial function and preserve skeletal muscle health during aging. 5-month-old SAMP8 mice, in a presarcopenia phase, have been randomly divided into 4 groups (n = 10): untreated controls and mice treated for two months with either physical exercise (0.5 km/h, on a 5% inclination, for 30 min, 5/7 days per week), ubiquinol 10 (500 mg/kg/day), or a combination of exercise and ubiquinol. Two months of physical exercise significantly increased mitochondrial damage in the muscles of exercised mice when compared to controls. On the contrary, ubiquinol and physical exercise combination significantly improved the overall status of the skeletal muscle, preserving mitochondrial ultrastructure and limiting mitochondrial depolarization induced by physical exercise alone. Accordingly, combination treatment while promoting mitochondrial biogenesis lowered autophagy and caspase 3-dependent apoptosis. In conclusion, the present study shows that ubiquinol supplementation counteracts the deleterious effects of physical exercise-derived ROS improving mitochondrial functionality in an oxidative stress model, such as SAMP8 in the presarcopenia phase.

Improvement of natural killer cell activity by in vitro active lipids (AL 721) administration in old mice.

Results of several experiments have given rise to the hypothesis that the decline of the immunocompetence with aging is at least in part related to alterations of the lipid membrane composition and, consequently, to a decrease in membrane fluidity. The age-dependent decline of mitogen responsiveness can, in fact, be reversed by a special lipid mixture designated as active lipids (AL 721), which acts by means of its fluidizing action on the plasma membrane. The purpose of this study was to examine the possibility of raising the low endogenous levels of Natural Killer (NK) activity by in vitro AL administration in old mice. When spleen cells from old mice were incubated in vitro with AL, a significant increase in cytotoxic activity was obtained over control cultures, without reaching, however, the levels observed in young mice. In spleen cells from young mice, the AL administration causes a slight augment of NK basal activity. These results suggest that cell membrane fluidity plays an important role in the efficiency of NK cells, giving support to the hypothesis that a rectification of rigidified cell membranes may represent a valuable approach to restore proper physiological functions in old age.

Effect of melatonin and pineal grafting on thymocyte apoptosis in aging mice.

We have evaluated the effect of chronic melatonin (MEL) treatment or pineal grafting (PG) in old mice on the apoptosis of both thymocytes and spleen lymphocytes under conditions of either serum deprivation or glucocorticoid or zinc administration. The apoptosis was correlated with the modulation of thymus and adrenal weight and corticosterone and zinc plasma levels induced by MEL treatment or PG in old mice. Balb/c mice (17-18 months old) were given supplements of MEL (40-50 micrograms/day/mouse) or grafted with a young pineal gland and then sacrificed after 8 months. Both the MEL treatment and PG partially prevented thymic involution in very old mice. Both treatments protected the thymic and spleen lymphocytes in old mice from the apoptosis induced by serum deprivation and recovered the reduced thymocyte sensitivity to the apoptosis induced by dexamethasone (DEX), present in old untreated animals, to the values found in young mice. DEX caused a bigger loss of G D /G 1 phase cells in MEL treated mice than in old untreated mice. The protective action of MEL treatment or PG on serum deprivation induced apoptosis was correlated with increased thymus weight, reduced adrenal weight and corticosterone levels and increased zinc plasma levels. The greater DEX-induced apoptosis found in MEL treated and PG mice was correlated with reduced adrenal weight and function. In vitro MEL did not affect thymocyte apoptosis in young or old mice. These results suggest that MEL treatment or PG prevent age-related thymus involution through regulation of thymocyte apoptosis which, in turn, occurs through modulation of the pituitary-adrenal axis and zinc turnover determined by the pineal hormone.

Adjuvant effect of low-dose interleukin-2 on antibody response to influenza virus vaccination in healthy elderly subjects.

It is well known that immune efficiency is frequently deteriorated in elderly people. The age-diminished antibody response to T-cell dependent antigens, such as influenza virus antigens, may explain the low protection offered by influenza vaccination in the elderly population. To investigate the possibility of increasing the antibody response to influenza virus vaccinations, we have conducted a nursing home-based study on the efficacy of IL-2. Seventy-five institutionalized elderly subjects (82 +/- 8 years) were enrolled in the study in the course of winter season 1991-1992. Thirty-nine subjects were treated with three subcutaneous daily injections of interleukin-2 (IL-2, 1 x 10(6) I.U./day) before vaccination and their antibody response was compared to that of 36 aged people receiving the vaccine only. An increased antibody response against influenza virus was present in vaccine plus IL-2 treated subjects (P < 0.001) but not in subjects treated with vaccine only. The number of protected subjects 45 days after vaccination was increased only in the IL-2-treated group (P = 0.045). The low-dose of IL-2 administered and the short-term treatment allowed a good tolerance to the IL-2 injection. In conclusion, the low-dose IL-2 treatment represents an effective means of inducing antibody response to influenza virus antigens in elderly subjects without appreciable toxicity.

Optimization of cytotoxic assay by target cell retention of the fluorescent dye carboxyfluorescein diacetate (CFDA) and comparison with conventional 51CR release assay.

This paper describes the comparison between a fluorimetric NK assay based on the target cell retention of fluorescent dye carboxyfluorescein diacetate (cFDA) and standard 51Cr release assay. The results provide several suggestions to improve the cytotoxic assay based on the use of the fluorogenic substrate showing that the measurements of cFDA retained by target cells represent a method of evaluating cytotoxicity completely comparable to the 51Cr release assay.

Plasticity of neuroendocrine-thymus interactions during aging.

Thymic regrowth and reactivation of thymic endocrine activity may be achieved even in old animals by different endocrinological or nutritional manipulations such as, (a) intrathymic transplantation of pineal gland or treatment with melatonin, (b) implantation of a growth hormone (GH) secreting tumor cell line or treatment with exogenous GH, (c) castration or treatment with exogenous luteinizing hormone-releasing hormone (LH-RH), (d) treatment with exogenous thyroxine or triiodothyronine, and (e) nutritional interventions such as arginine or zinc supplementation. These data strongly suggest that thymic, involution is a phenomenon secondary to age-related alterations in neuroendocrine-thymus interactions and that it is the disruption of such interactions in old age that is responsible for age-associated dysfunction. With regard to the mechanisms involved in hormone-induced thymic reconstitution, it is at present, difficult to draw any definitive conclusions. The effect of GH, thyroid hormones, and LH-RH may be due to the presence on thymic epithelial cells supposed to produce thymic peptides, of the specific hormone receptors. Melatonin or other pineal factors may also act through specific receptors, but experimental evidence is still lacking. The role of zinc, whose turnover is usually reduced in old age, is diverse. The effects range from the reactivation of zinc-dependent enzymes, required for both cell proliferation and apoptosis, to the reactivation of thymulin, a zinc-dependent thymic hormone. The role of zinc may even be more crucial. According to recent preliminary data obtained both in animal and human studies, it appears that the above reported endocrinological manipulations capable of restoring thymic activity in old age, may act also by normalizing the altered zinc pool.

Timing of appearance and disappearance of IFN and IL-2 induced natural immunity during ontogenetic development and aging.

The age-dependent modifications of both basal and lymphokine-induced natural killer (NK) activity were analyzed in Balb/c inbred mice by measuring either the percentage of specific lytic activity in a 51 Cr release assay or the percentage of cells capable of binding the YAC-1 target cells. The basal lytic activity of spleen cells is low at birth, then it increases progressively and reaches a peak between 5 and 8 weeks of age and decreases thereafter, displaying in 25-month-old mice only 10% of the NK activity found in young mice. The number of spleen cells capable of binding target cells is higher at birth and then it declines progressively-old mice show about 40% of the binding capacity found in young mice. Significant in vitro NK activation is obtained in spleen cells from 25-, 50- and 750-day-old mice by incubation with interleukin-2 (IL-2) (1.8-fold increase in 25- and 50-day-old mice and 2.6-fold increase in 750-day-old mice) while no effect is obtained in 15-day-old mice. The responsiveness to in vitro stimulation with interferon (IFN) is not present in 15-day-old mice, however it appears in a defective way at the age of 25 days, and reaches its highest level in 50- and 180-day-old mice, (3.5- and 4.5-fold increase), still remaining present in 630-day-old mice, (two-fold increase) but not in 750-day-old mice. The in vivo injection with IFN increases the spleen cell NK activity of 25- and 50-day-old mice (3.6- and 2.3-fold increase respectively) but not of 15- and 750-day-old mice. The present data here confirm the existence of age-dependent variations of spleen cell NK activity and suggest a similar sequential timing of appearance/disappearance of IL-2 and IFN responsive spleen cells during ontogenetic development and aging respectively.

Immune enhancement by conditioning of senescent mice. Comparison of old and young mice in learning ability and in ability to increase natural killer cell activity and other host defense reactions in response to a conditioned stimulus.

It has been clearly demonstrated that immune responses may be conditioned in a manner similar to that of the classical Pavlovian experiments. Evidence of impaired immune function in aging has raised the question of whether psychological conditioning of an immune response can also be effective in old age. The knowledge that aged mice have decreased spleen cell natural killer (NK) activity and that NK cytotoxicity, at least in young mice, can be psychologically conditioned led us to explore in old mice the possibility of conditioning the response of NK cell activity using the odor of camphor as the conditioned stimulus (CS) and the injection of Poly I:C as the unconditioned stimulus (US). Young and old male mice were divided into five and six groups, respectively. They received the CS and/or the US in association (conditioning) trials (sessions 1-9). Mice were exposed to the camphor odor alone at 72 hours after the final association trial to observe the conditioning phenomenon (session 10). The group conditioned with Poly I:C and camphor and receiving the CS at session 10 showed statistically significant increases in spleen cell NK activity over those of the control groups that did not receive the CS treatment at session 10 (2.6- and 4.0-fold increase in young and old, respectively). Treatment with camphor odor alone had no effect on boosting NK cell activity. These findings demonstrate the possibility of conditioning immune responses in old age, offering a valuable tool for attenuating age-related immune deterioration in various species, including the human. In addition, these results again confirm highly significant immune enhancement by classical conditioning and extend previous findings from female mice to males as well.

Natural killer cell activity in endometriosis: correlation between serum estradiol levels and cytotoxicity.

OBJECTIVE: To evaluate the correlation between natural killer cell activity and serum estradiol (E2) levels in patients with different stages of endometriosis. METHODS: Natural killer cell activity of peripheral blood lymphocytes and serum E2 levels were evaluated in 73 women who underwent laparoscopy for pelvic pain, infertility, and benign adnexal masses. RESULTS: The 33 patients (45%) with endometriosis showed a significant decrease in natural killer cell activity in relationship to an increase in disease stage (correlation coefficient r = -0.83, P < .001). A significant inverse relationship was observed between cytotoxicity and serum E2 levels (correlation coefficient r = -0.89, P < .001). CONCLUSION: The relationship between natural killer cell activity and serum E2 levels suggests that an immunoendocrine interaction plays an important role in the progression of endometriosis.

Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release.

OBJECTIVE: To investigate the effect of disease on peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cell cytotoxicity and to provide additional information concerning the cell-mediated immune function in endometriosis. METHODS: Chemotactic index of peripheral blood polymorphonuclear leukocytes, natural killer cell activity, and plasma estradiol (E2) and plasma prostaglandin (PG) E2 levels were evaluated in 46 women who underwent laparoscopy or laparotomy for pelvic pain, infertility, and/or benign adnexal masses. RESULTS: The 20 women (43%) with endometriosis showed a decrease in peripheral blood polymorphonuclear leukocyte chemotactic index, related to advanced disease stage (P < .001). A significant inverse correlation was observed between plasma PGE2 levels and chemotactic index in stage III and IV endometriosis (r = -.73, P = .004). Similarly, natural cytotoxicity was decreased significantly with respect to the stage of endometriosis (P = .004) and related inversely to plasma PGE2 levels (r = -.74, P = .003). A direct relationship was observed between PGE2 and plasma E2 levels (r = .59, P = .006). CONCLUSION: Advanced endometriosis is associated with decreased peripheral blood polymorphonuclear leukocyte chemotactic index and natural killer cytotoxicity, which may be related to plasma PGE2 and E2 levels.

Natural cytotoxicity and GnRH agonist administration in advanced endometriosis: positive modulation on natural killer activity.

OBJECTIVE: To investigate the effects of pharmacologic suppression of ovarian function on the immune system, with respect to the clinical outcome of endometriosis and the possibility of an immunoendocrine combined treatment. METHODS: After informed consent, 25 of 37 patients with revised American Fertility Society stage III and IV endometriosis who underwent postoperative medical treatment were selected and enrolled for this immunoendocrine longitudinal study. Medical treatment consisted of tryptorelinum depot injection, 3.75 mg/month for 24 weeks. Blood samples were collected before the first injection in the early follicular phase, day 2-3 of the cycle, and during medical treatment (every 4 weeks) and follow-up (every 6 months). At the end of the study, we had ten blood samples per patient to evaluate the cytotoxic activity, the number of natural killer cells, and the serum levels of estradiol. Natural killer activity was determined against the K562 cell line by target cell retention of the fluorescent dye carboxyfluorescein diacetate. RESULTS: A positive immunomodulating effect was observed during GnRH agonist administration. In particular, a significant progressive increase in natural killer cell activity was defined within the first 12 weeks of medical treatment; after three injections, we observed the highest values of cytotoxicity, with a median of 7.1 lytic units (range 0.3-14.0; P = .02). Natural cytotoxicity then decreased toward a plateau, which persisted during therapy completation and follow-up, with slight fluctuations. In patients who had recurrence, the values of natural killer cell activity were constantly lower than those in patients with disease-free follow-up, particularly within the first 12 weeks of medical treatment.

Induction of natural killer cell activity and perforin and granzyme B gene expression following continuous culture of short pulse with interleukin-12 in young and old mice.

Anticancer immunotherapy with cytokines is often limited by the occurrence of severe toxicity, particularly in older age groups, which are characterized by a reduced tolerance to antineoplastic therapies. We, and others, have recently demonstrated the efficacy of pulsing procedures with IL-2 as a new therapeutic strategy to induce antitumor cytotoxic cells. The aim of this paper was to evaluate the effect of IL-12 on NK cell activity in young and old mice and to investigate the possibility of inducing NK cytotoxicity and perforin and granzyme B gene expression through a brief exposure of spleen lymphocytes from young and old mice to IL-12. Pulsed lymphocytes were compared with non-pulsed cells cultured continuously in IL-12. IL-12 was able to boost both endogenous and IL-2-induced NK cell activity in young and old mice; the levels of cytotoxicity were lower in old than in young animals although the relative increase of IL-12 plus IL-2 versus IL-2 alone was greater for old mice. Comparable levels of NK cell activity were obtained in pulsed (5 min-1 hour) and non-pulsed lymphocytes from both young and old mice after one or three days of culture. The efficacy of the pulsing procedure was evident in both endogenous and IL-2-induced NK cytotoxicity. The mRNA encoding perforin and granzyme B were markedly and similarly enhanced in both IL-12-pulsed and non-pulsed lymphocytes in comparison with control cells. The results demonstrate the effectiveness of IL-12 pulsing in inducing antitumor cytotoxic cells, suggesting the possibility of using IL-12 pulsing, alone or in combination with IL-2, in the immunotherapy of both young and old subjects.