Placental mRNA Expression of Neurokinin B Is Increased in PCOS Pregnancies with Female Offspring.

Current research suggests that polycystic ovary syndrome (PCOS) might originate in utero and implicates the placenta in its pathogenesis. Kisspeptin (KISS1) and neurokinin B (NKB) are produced by the placenta in high amounts, and they have been implicated in several pregnancy complications associated with placental dysfunction. However, their placental expression has not been studied in PCOS. We isolated mRNA after delivery from the placentae of 31 PCOS and 37 control women with term, uncomplicated, singleton pregnancies. The expression of KISS1, NKB, and neurokinin receptors 1, 2, and 3 was analyzed with real-time polymerase chain reaction, using ß-actin as the reference gene. Maternal serum and umbilical cord levels of total testosterone, sex hormone-binding globulin (SHBG), free androgen index (FAI), androstenedione, dehydroepiandrosterone sulfate (DHEAS), Anti-Mullerian hormone (AMH), and estradiol were also assessed. NKB placental mRNA expression was higher in PCOS women versus controls in pregnancies with female offspring. NKB expression depended on fetal gender, being higher in pregnancies with male fetuses, regardless of PCOS. NKB was positively correlated with umbilical cord FAI and AMH, and KISS1 was positively correlated with cord testosterone and FAI; there was also a strong positive correlation between NKB and KISS1 expression. Women with PCOS had higher serum AMH and FAI and lower SHBG than controls. Our findings indicate that NKB might be involved in the PCOS-related placental dysfunction and warrant further investigation. Studies assessing the placental expression of NKB should take fetal gender into consideration.

ACTH vs steroids for the treatment of acute gout in hospitalized patients: a randomized, open label, comparative study.

The management of acute gout in the hospital setting may be challenging since most patients are elderly with multiple unstable comorbidities. However, there are no prospective clinical trials for hospitalized patients with gout to guide optimal management. Evidence indicates that steroids or adrenocorticotropic hormone (ACTH) may be effective and safe therapeutic options for these patients. This study aimed at directly comparing the efficacy and safety of ACTH vs betamethasone for the treatment of gout in hospitalized patients. This is the first prospective clinical trial for hospitalized patients with gout. We designed a randomized, open label study to assess the efficacy and safety of a single intramuscular injection of either ACTH or betamethasone in hospitalized patients with acute gout. Primary efficacy endpoints were the change in intensity of pain as recorded using a Visual Analogue Scale (VAS) at baseline compared to 24 h (ΔVAS24h), and 48 h. Moreover, we assessed safety and effects on the hypothalamic-pituitary-adrenal (HPA) axis, glucose and lipid homeostasis, bone metabolism, electrolytes and renal function. 38 patients were recruited. Both treatments were highly effective. The mean ± SE ΔVAS24h and ΔVAS48h for ACTH was 4.48 ± 0.29 and 5.58 ± 0.26, respectively. The mean ± SE ΔVAS24h and ΔVAS48h for betamethasone was 4.67 ± 0.32 and 5.67 ± 0.28, respectively. Direct comparison between the two groups at 24 h and 48 h did not show statistically significant differences. Both treatments were well tolerated and safe. The effects on all metabolic parameters were mostly minimal and transient for both treatments. However, ACTH may affect less the HPA axis and bone metabolism compared to betamethasone, thus leading to the conclusion that. ACTH and betamethasone are effective and safe for the management of acute gout in hospitalized patients but that ACTH may associate with less disturbance of the HPA axis and bone metabolism. Our data support the use of both drugs as first line treatments for hospitalized patients with gout.Clinical trial registration: ClinicalTrials.gov NCT04306653.

Differences in thyroid function tests after administration of a single supraphysiological dose of ACTH or a high dose of a synthetic steroid.

OBJECTIVE: Although the suppressive action of synthetic steroids on the hypothalamus-pituitary-thyroid (HPT) axis is established, little is known regarding the effect of the administration of synthetic adrenocorticotropic hormone (ACTH). DESIGN: In the context of a randomized, open label, comparative study assessing the efficacy and safety of ACTH and betamethasone in the treatment of hospitalized patients with acute gout, we compared the effects of these agents on thyroid function tests. METHODS: Serum TSH, total T4 and T3 and cortisol were measured before and at 24 and 48 h after a single intramuscular dose of synthetic ACTH (1 mg) or betamethasone (6 mg), in 38 hospitalized patients with acute gout and normal thyroid function. RESULTS: The final analysis included 32 patients, due to missing data. The ACTH and betamethasone groups did not differ regarding the mean age, gender, severity of gout attack, and baseline thyroid parameters. In the ACTH group TSH and T4 were significantly decreased at 24 and at 48 h compared to baseline, while T3 was decreased at 24 but not at 48 h. In the betamethasone group T3 remained stable; TSH and T4 decreased significantly from baseline levels at 24 h; at 48 h, TSH had returned to and T4 showed a partial rebound towards pre-treatment values. CONCLUSIONS: A single IM administration of 1 mg of synthetic ACTH has more profound and prolonged effects on the HPT axis, lasting for at least 48 h, compared to a single IM dose of 6 mg betamethasone.

Benign thyroid nodules respond to a single administration of 0.3mg recombinant human thyrotropin with highly variable volume increase.

Introduction: The nature of thyroid nodules is heterogenous. Most of them are benign and, in the absence of pressure symptoms of adjunct structures, no treatment is needed. Our objective was to investigate the acute effects of a low dose of recombinant human TSH (rhTSH) on the volume of benign thyroid nodules. Methods: we studied 27 nodules (14 isoechoic and 13 hypoechoic) in 15 (11 women and 4 men; mean age: 51.0 ± 15.9 years) consecutive patients with one to three well-separated asymptomatic benign thyroid nodules. All subjects were euthyroid, with negative thyroid antibodies, and none received levothyroxine. The total thyroid volume and thyroid nodule volume were sonographically determined by two independent examiners (P.B. and M.M.) before, 48 hours and 6 months post intramuscular (IM) administration of 0.3mg rhTSH, and the mean values of the two examiners' measurements were used; thyroid function tests were obtained at the same time points. Results: The mean volume of isoechoic nodules increased by 57.3%, of hypoechoic nodules by 46.6% and of the surrounding thyroid parenchyma by 70.4% 48 hours post-rhTSH; mean volumes had returned to baseline levels 6 months later. A large variance in the volume change responses was observed. The relative change in nodule volume (defined as the percent change in nodule volume divided by the percent change in the surrounding parenchyma) from baseline to 48 hours was significantly higher in isoechoic versus hypoechoic nodules (p<0.05). Conclusions: A single dose of 0.3 mg rhTSH transiently increased the volume of benign thyroid nodules. The increase was more pronounced in isoechoic nodules and had a great variability. Our findings could be useful in the management of benign thyroid nodules, by helping in understanding which nodules would be more responsive to TSH suppression therapy.

Therapeutic Equivalence of a New Preparation of Liquid Levothyroxine with Tablets in Patients with Overt Primary Hypothyroidism.

BACKGROUND: A new liquid levothyroxine (LT4) dissolved in glycerol and water has recently been developed by a Greek pharmaceutical company (Uni-Pharma, Athens, Greece). OBJECTIVES: To evaluate the therapeutic equivalence of this new liquid LT4 preparation versus the already existing tablet formulation of the same manufacturer, in order to obtain approval by the Greek National Organization for Medicines. METHODS: This was a prospective, randomized, cross-over phase III study. The study included 50 patients (9 men and 41 non-pregnant women, with a mean age of 42.5 ± 12.5 years), with documented overt primary hypothyroidism. All subjects were well controlled on substitution therapy with various LT4 formulations. None of the patients had known LT4 malabsorption. The patients were randomized into 2 groups (A and B). The individuals of group A initially received T4® tablets for 10 ± 2 weeks and subsequently switched to T4® drops (100 µg/mL solution) at the same dose for another 10 ± 2 weeks. In group B, the reverse procedure was followed. Total T3 (T3), free T4 (fT4), and TSH were measured in all participants at enrollment and at the end of each 10 ± 2-week trial period. RESULTS: Out of the 50 recruited patients, 6 were lost to follow-up and 5 were excluded due to non-compliance with the study protocol. In the 39 patients who completed the study, the serum TSH levels after 10 ± 2 weeks of treatment either with T4® tablets or with T4® drops did not differ (1.759 ± 1.104 vs. 2.076 ± 1.334 mIU/L, mean ± SD). CONCLUSIONS: In hypothyroid patients, the new liquid LT4 preparation (T4® drops) is therapeutically equivalent to the tablet form (T4® tablets).

Thyroid-stimulating hormone is not the primary regulator of thyroid development in euthyroid children and adolescents living in an iodine-replete area.

OBJECTIVES: It is known that there are multiple factors which can affect thyroid gland development during childhood and adolescence. Our aim was to investigate this issue by examining the relationships between age, sex, several anthropometric parameters, pubertal status, thyroid function tests, and iodine intake status with thyroid volume (TV) in children and adolescents. STUDY DESIGN: This was a cross-sectional field study conducted in 11 representative cities and villages of Uzbekistan. Six hundred and ten children and adolescents participated. Anthropometric indices and TV were estimated. In addition, thyroid function tests (TFTs) and urinary iodine excretion (UIE) measures were obtained. RESULTS: Median UIE was 151 µg/L, thus the studied areas were iodine-sufficient. TFTs fluctuated in both genders during childhood and adolescence and the thyroid growth spurt was observed, in both sexes, at the ages of 12 and 13 years, which coincided with the age of menarche in girls. Thyroid volume was positively correlated with body surface area (BSA) (r = 0.800, p < 0.001), age (r = 0.780, p < 0.001), fat-free mass (FFM) (r = 0.797, p < 0.001) and negatively correlated with serum TSH (r = -0.154, p = 0.05). No association between thyroid volume and UIE was observed. CONCLUSIONS: In euthyroid children and adolescents living in iodine-replete areas, thyroid gland development appears to follow the pattern of linear growth and displays a growth spurt at the onset of puberty, probably due to the abrupt increase of circulating sex steroids. At this age, TSH does not appear to be the main regulator of thyroid gland development.

No increase in renal iodine excretion during pregnancy: a telling comparison between pregnant women and their spouses.

OBJECTIVE: Adequate dietary iodine intake is necessary for normal thyroid gland function at all times, and most particularly during pregnancy. Increased iodine loss is cited, among other factors, as responsible for the increased iodine demand in this period. Our aim was to compare renal iodine excretion between women during all three pregnancy trimesters with that of their spouses and thereby to estimate the iodine intake in an a large sample of pregnant women in urban areas in Greece. DESIGN: Four hundred twenty-four healthy pregnant women were included prospectively (residents of Athens n=218, residents of Patras n=206). The spouses of 177 of these women following the same diet were also studied. Determinations included serum FT4, TSH and aTPO and urinary iodine excretion (UIE). RESULTS: No difference was found either in median UIE throughout pregnancy or between the UIE of the pregnant women and their spouses during the trimesters. Throughout pregnancy, mild iodine deficiency was noted and was classified as mild in 60%, moderate in 30% and severe in 10% of the women studied. Users of iodized salt had significantly higher median UIE compared with non-users. Serum FT4 levels decreased and TSH increased as pregnancy progressed. CONCLUSIONS: Our study indicates that renal iodine excretion is not increased during pregnancy. This finding needs to be confirmed by further investigation in other populations with different iodine intakes. Thus, increased iodine requirements in pregnancy are possibly due to extra-renal causes. The population of pregnant women in Greek urban areas is mildly-and often moderately and severely-iodopenic and needs to be treated accordingly.

Salivary adiponectin levels are associated with training intensity but not with bone mass or reproductive function in elite Rhythmic Gymnasts.

Elite Rhythmic Gymnasts (RGs) constitute a unique metabolic model and they are prone to developing Anorexia Athletica. The aim of the present study was to evaluate the effect of training intensity on salivary adiponectin levels and assess a possible role of salivary adiponectin levels as a predictive factor of reproductive dysfunction and bone mass acquisition in elite RGs. The study included 80 elite female RGs participating in the World Rhythmic Gymnastics Championship tournament held in Montpellier, France on September 2011. Anthropometric values were assessed, training data and menstrual pattern were recorded, bone mass was measured with Broadband ultrasound attenuation (dB/Mhz) and baseline salivary adiponectin levels were determined. The athletes were classified as intensely and very intensely trained, considering the mean training intensity (40.84h/week). Moreover, considering their reproductive status, they were divided into RG's with normal menstruation, primary amenorrhea and oligomenorrhea. All comparisons were adjusted to age, BMI and body fat percentage differences. Very intensely trained RGs showed higher salivary adiponectin levels (p=0.05). Moreover, salivary adiponectin levels showed significant correlation with training intensity (r=0.409, p=0.003). On the other hand, no association of salivary adiponectin levels was documented with either reproductive function or bone mass acquisition. The results of the present study suggest that, in elite RGs, salivary adiponectin levels are associated with the intensity of training, possibly reflecting the deterioration of energy balance rather than the training stress. On the other hand, a predictive role of salivary adiponectin levels in reproductive dysfunction or bone mass acquisition could not be supported.

The effect of prolonged aerobic exercise on serum adipokine levels during an ultra-marathon endurance race.

OBJECTIVE: To evaluate the effect of prolonged intensive aerobic exercise and acute energy deficit (180 km ultra-marathon race) on serum leptin, adiponectin, resistin and visfatin levels and their association and interaction with serum cortisol and insulin levels in highly trained ultra-endurance runners. DESIGN: The study included 17 highly trained ultra-endurance male athletes (mean age 51.29±6.84 years and body mass index (ΒΜΙ) 23.51±1.90) participating in the 5th Olympian Race held in Greece on May 2010. Anthropometric values were assessed; Serum cortisol, insulin, leptin, adiponectin, resistin and visfatin levels were measured at baseline, post-exercise and ~20 hours after the end of the race. RESULTS: All hormonal values of the post-exercise and recovery status were corrected for plasma volume changes. The estimated energy deficit during the ultra-endurance event was about 5000 Kcal. At the end of the race serum resistin levels were elevated (p<0.001) and serum leptin levels were reduced (p<0.001) and failed to reach pre-exercise levels, although showing a tendency towards restoration. No significant changes were noted in serum adiponectin and visfatin levels. CONCLUSIONS: Ultra-endurance aerobic exercise and acute negative energy balance lead to an up-regulation of serum resistin levels and a down-regulation of serum leptin levels.

Improved levothyroxine pharmacokinetics after bariatric surgery.

BACKGROUND: The absorption of levothyroxine (LT4) is affected by many factors. Bariatric surgery is recommended in severely obese patients. The aim of this study was to determine the consequences of bariatric surgery on LT4 pharmacokinetic parameters, and to identify the regions of the gastrointestinal tract where LT4 is absorbed in patients with severe obesity before and after surgery. METHODS: We studied 32 severely obese nonhypothyroid patients who underwent sleeve gastrectomy (SG; n=10), Roux-en-Y gastric bypass (RYGBP; n=7), or biliopancreatic diversion with long limbs (BPD-LL; n=15). Before surgery, from 8:00 a.m., blood samples were collected before and every 30 minutes after the oral administration of a solution of 600 µg of LT4. The same procedure was repeated 35 days after surgery. We estimated the pharmacokinetic parameters of LT4 before and after surgery, including the area under the curve (AUC), the peak thyroxine concentration (Cmax), and the time to peak thyroxine concentration (Tmax). RESULTS: Following surgery, in the SG group, the mean AUC was higher than it was before surgery (18.97±6.01 vs. 25.048±6.47 [µg/dL]·h; p<0.01), whereas the values of Cmax and Tmax were similar to those before surgery. In the RYGBP group, mean AUC, Cmax, and Tmax were similar before and after surgery. In the BPD-LL group, mean AUC and Cmax were higher after surgery than before (14.18±5.64 vs. 25.51±9.1 [µg/dL]·h, p<0.001; 5.62±1.34 vs. 8.16±2.57 µg/dL, p<0.001, respectively), whereas Tmax was similar. CONCLUSIONS: The pharmacokinetic parameters of LT4 absorption are improved following SG and BPD-LL types of bariatric procedures. We conclude that the stomach, the duodenum, and the upper part of the jejunum are not sites for LT4 absorption, because in the above-mentioned bariatric procedures these are bypassed or removed.

Diet, physical exercise and Orlistat administration increase serum anti-Müllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS).

The present study investigates the combined effect of diet, physical exercise and Orlistat for 24 weeks, on serum anti-Müllerian hormone (AMH) levels in overweight and obese women with polycystic ovary syndrome (PCOS) and in overweight and obese controls. Sixty-one (61) selected women with PCOS and 20 overweight and obese controls followed an energy-restricted diet, physical exercise plus Orlistat administration (120 mg, 3 times per day) for 24 weeks. At baseline, week 12 and week 24, serum levels of AMH, FSH, LH, PRL, androgens, sex hormone-binding globulin (SHBG), glucose, and insulin were measured and Free Androgen Index (FAI) and Insulin Resistance (IR) indices were calculated. In PCOS women, serum AMH levels increased after 12 and 24 weeks of treatment. After 12 weeks LH and SHBG were increased, while Testosterone decreased. After 12 and 24 weeks, FAI was decreased and all indices of IR were significantly improved. We concluded that in overweight and obese women with PCOS Orlistat administration, combined with diet and physical exercise, for 24 weeks, resulted in significant weight loss, improvement of hyperandrogenism and insulin sensitivity, and increased serum AMH levels.

Measurement of salivary resistin, visfatin and adiponectin levels.

Hormonal determination in saliva offers several advantages. Peptides enter the salivary glands either by active transport mechanisms or are expressed and secreted by the salivary glands themselves. The collection of saliva is a noninvasive, easily repeatable and less stressful technique than blood withdrawal. The purpose of the present study was to introduce a method for measuring salivary resistin, visfatin and adiponectin levels and to evaluate their associations with serum levels. Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for serum with minor modifications. The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary levels with serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. To our knowledge, this is the first study to report the determination of resistin and visfatin in saliva and the significant correlation of salivary resistin with serum levels, while it confirmed the significant association between salivary and serum adiponectin. The introduction of salivary determinations of adipokines could contribute to the elucidation of the physiology and the role of the specific adipokines in various clinical conditions (obesity, insulin resistance, inflammation, reproduction, energy imbalance and stress response).

Impaired pharmacokinetics of levothyroxine in severely obese volunteers.

BACKGROUND: Suppressive or replacement doses of levothyroxine (LT4) are affected by the rate and extent of the active ingredient absorbed, as well as by the lean body mass. Obesity has reached epidemic proportions worldwide and is related with many comorbidities. The aim of this study was to determine the pharmacokinetic parameters of LT4 in severely obese individuals and compared them with similar data in lean control subjects. METHODS: We studied 62 euthyroid subjects who had negative tests for anti-thyroid peroxidise antibodies (Ab-TPO). Thirty eight of these subjects were severely obese but otherwise healthy (severe obese subjects [SOS] group). Twenty-four were healthy control subjects (control group), with a body mass index of 23.3 ± 1.7 kg/m(2). Subjects received 600 µg oral sodium LT4 after an overnight fast. Serum triiodothyronine (T3), T4, and thyroid-stimulating hormone were measured at baseline. Serum T4 and T3 was measured 0.5, 1, 1.5, 2, 2.5, 3, and 4 hours after LT4 administration. RESULTS: Baseline serum T4 and thyroid-stimulating hormone concentrations were higher in the SOS group than in the control group; serum T3 was similar in the two groups. The corrected area under the curve and the maximum T4 concentration after LT4 administration were lower, whereas the time to maximum concentration from the baseline was higher in SOS than in the control group. The estimated plasma volume was higher in the SOS than in the control group. Mean serum T3 levels increased gradually during the four hours after LT4 administration in the control group. In contrast, they decreased gradually in the SOS group. CONCLUSIONS: Severely obese individuals may need higher LT4 suppressive or replacement doses than normal-weight individuals due, among other factors, to impaired LT4 pharmacokinetic parameters. The latter could be attributed to their higher plasma volume and/or to delayed gastrointestinal LT4 absorption. T4 conversion to T3 might be defective in severe obesity.

A beta integrin subunit regulates bacterial phagocytosis in medfly haemocytes.

We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.

Elk-1 associates with FAK, regulates the expression of FAK and MAP kinases as well as apoptosis in HK-2 cells.

Focal adhesion kinase (FAK), MAP kinases and the nuclear transcription factor Elk-1 have been reported to be implicated in the same cellular processes, however, their direct or indirect interaction and potential function(s) has not been documented. Here, we explored the association of FAK with Elk-1, the implication of Elk-1 in the regulation of FAK and MAP kinases expression as well as apoptosis, in HK-2 cells. Biochemical and immunofluorescence approaches strongly support the association of low molecular weight protein bands, recognized by FAK antibodies, with Elk-1 or p(ser383)Elk-1. The FAK/Elk-1 complex is found, mainly, in the cytoplasm, near the nuclear membrane periphery, raising the possibility that Elk-1 may have alternative extranuclear function(s) in HK-2 cells. Furthermore, we demonstrated that Elk-1 siRNA-mediated knockdown experiments, increased apoptosis. By contrast, Elk-1 siRNA decreased significantly the expression of FAK and MAP kinases, supporting the hypothesis that Elk-1 may act as a potential physiological substrate and regulator of FAK and MAP kinases expression. These results strongly support that Elk-1 protein is a novel binding-protein partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk-1.

Elk-1 is a novel protein-binding partner for FAK, regulating phagocytosis in medfly hemocytes.

Focal adhesion kinase (FAK) and its downstream signaling targets, mitogen-activated protein kinase (MAPKs), are implicated in the process of phagocytosis by insect hemocytes. The goal of this study was to explore further the signaling pathways underlining the process of phagocytosis. The combination of bioinformatics, biochemical, and immunofluorescence approaches strongly support the expression of Elk-1-like protein in medfly hemocytes. Elk-1 is phosphorylated in E. coli or latex beads-challenged hemocytes and osmotic loading experiments as well as flow cytometry analysis demonstrated that Elk-1-like protein regulates the uptake of bacteria. RNA interference (RNAi) and pharmacological inhibitors show that the signaling for Elk-1 phosphorylation is transmitted via FAK/Src and MAPKs pathways. Furthermore, confocal analysis clearly shows that FAK and the phosphorylated FAK at Y397 are localized in the nucleus and cytoplasm, whereas, the phosphorylated Elk-1-like protein is exclusively localized in the nucleus. Finally, co-immunoprecipitation and reciprocal co-immunoprecipitation analysis demonstrated the association of low molecular weight protein bands recognized by FAK antibodies, with Elk-1 or phospho-Elk-1 at ser 383 and confocal microscopy specifies that this association occurs only in the nucleus. These results are strongly supporting that Elk-1-like protein is a novel protein-binding partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk-1 generally. Evidently, the complex participates in the process of phagocytosis in medfly hemocytes.

Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes.

In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling.

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.