Marie Rubér

Different cytokine profiles in patients with a history of gangrenous or phlegmonous appendicitis

Appendicitis is one of the most common and costly acute abdominal states of illnesses. Previous studies suggest two types of appendicitis which may be different entities, one which may resolve spontaneously and another that progresses to gangrene and perforation. Gangrenous appendicitis has a positive association to states of Th1 mediated immunity whereas Th2 associated immune states are associated with lower risk of appendicitis. This study investigated the inflammatory response pattern in patients previously appendicectomized for gangrenous (n = 7), or phlegmonous appendicitis (n = 8) and those with a non‐inflamed appendix (n = 5). Peripheral blood mononuclear cells were analysed with ELISPOT analysis for number of spontaneous or antigen/mitogen stimulated IFN‐γ, IL‐4, IL‐10 and IL‐12 secreting cells or with ELISA for concentration of spontaneous or antigen/mitogen stimulated IFN‐γ, IL‐5 and IL‐10. Spontaneously IL‐10 secreting cells/100 000 lymphocytes were increased in the gangrenous group compared to the phlegmonous group (P = 0·015). The median concentration of IL‐10 secreted after Tetanus toxoid (TT)‐stimulation were higher in the gangrenous group and the control group, than the phlegmonous group (P = 0·048 and P = 0·027, respectively). The median concentration of TT induced IFN‐γ secretion was higher for the gangrenous group compared to both the phlegmonous group and the control group (P = 0·037 and P = 0·003). Individuals with a history of gangrenous appendicitis demonstrated ability to increased IL‐10 and IFN‐γ production. The increased IFN‐γ may support the notion of gangrenous appendicitis as an uncontrolled Th1 mediated inflammatory response and increased IL‐10 may speculatively indicate the involvement of cytotoxic cells in the progression to perforation.

Systemic Th17-like cytokine pattern in gangrenous appendicitis but not in phlegmonous appendicitis

BACKGROUND Increasing circumstantial evidence suggests that not all patients with appendicitis will progress to perforation and that appendicitis that resolves may be a common event. Based on this theory and on indications of aberrant regulation of inflammation in gangrenous appendicitis, we hypothesized that phlegmonous and gangrenous appendicitis are different entities with divergent immunoregulation. METHODS Blood samples were collected from patients with gangrenous appendicitis (n = 16), phlegmonous appendicitis (n = 21), and nonspecific abdominal pain (n = 42). Using multiplex bead arrays, we analyzed a range of inflammatory markers, such as interleukin (IL)-1ra, IL-1rbeta, IL-2, IL-6, IL-10, IL-12p70, IL-15, and IL-17; interferon-gamma; tumor necrosis factor; CXCL8; CCL2; CCL3; and matrix metalloproteinase (MMP)-1 MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MMP-13 in blood. RESULTS Compared with patients with phlegmonous appendicitis and nonspecific abdominal pain, the patients with gangrenous appendicitis had increased levels of the proinflammatory markers IL-6, CCL2, IL-17, MMP-8, and MMP-9 (P < or = .04 each) accompanied by increased levels of the anti-inflammatory cytokines IL-1ra and IL-10 (P < or = .02). Patients with phlegmonous appendicitis had increased levels of IL-10 only. CONCLUSION The finding of a pattern of inflammatory markers compatible with the highly inflammatory Th17 subset in sera from patients with gangrenous appendicitis, but not in phlegmonous appendicitis, supports the hypothesis that gangrenous and phlegmonous appendicitis are different entities with divergent immune regulation. Additional studies of the differential immunopathogenesis of phlegmonous and gangrenous appendicitis are warranted, as this may have important implications in the diagnosis and management of patients with suspicion of appendicitis.

Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates.

This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON‐1, extracellular) and type 2 (PON‐2, intracellular) in boar semen. To evaluate PON‐1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON‐1 and total cholesterol levels. Seminal‐plasma PON‐1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal‐plasma PON‐1 concentration and enzymatic activity were negatively correlated (r = −0.763; P < 0.01). The activity of seminal‐plasma PON‐1 negatively correlated with ejaculate volume (r = −0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal‐plasma cholesterol concentration positively correlated with PON‐1 activity (r = 0.773; P < 0.01), but negatively correlated with PON‐1 concentration (r = −0.709; P < 0.05). The presence of intracellular PON‐2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON‐2 localised to the post‐acrosomal area of the sperm head and principal piece of the tail in membrane‐intact spermatozoa. In summary, PON is present in boar semen, with PON‐1 at low levels in seminal plasma and PON‐2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal‐plasma parameters. Mol. Reprod. Dev. 82: 58–65, 2015.

The Seminal Plasma of the Boar is Rich in Cytokines, with Significant Individual and Intra‐Ejaculate Variation

The boar, as human, sequentially ejaculates sperm‐rich and sperm‐poor fractions. Seminal plasma (SP) spermadhesins (PSP‐I/PSP‐II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility.

Selection for higher fertility reflects in the seminal fluid proteome of modern domestic chicken.

The high egg-laying capacity of the modern domestic chicken (i.e. White Leghorn, WL) has arisen from the low egg-laying ancestor Red Junglefowl (RJF) via continuous trait selection and breeding. To investigate whether this long-term selection impacted the seminal fluid (SF)-proteome, 2DE electrophoresis-based proteomic analyses and immunoassays were conducted to map SF-proteins/cytokines in RJF, WL and a 9th generation Advanced Intercross Line (AIL) of RJF/WL-L13, including individual SF (n=4, from each RJF, WL and AIL groups) and pools of the SF from 15 males of each group, analyzed by 2DE to determine their degree of intra-group (AIL, WL, and RJF) variability using Principal Component Analysis (PCA); respectively an inter-breed comparative analysis of intergroup fold change of specific SF protein spots intensity between breeds. The PCA clearly highlighted a clear intra-group similarity among individual roosters as well as a clear inter-group variability (e.g. between RJF, WL and AIL) validating the use of pools to minimize confounding individual variation. Protein expression varied considerably for processes related to sperm motility, nutrition, transport and survival in the female, including signaling towards immunomodulation. The major conserved SF-proteins were serum albumin and ovotransferrin. Aspartate aminotransferase, annexin A5, arginosuccinate synthase, glutathione S-transferase 2 and l-lactate dehydrogenase-A were RJF-specific. Glyceraldehyde-3-phosphate dehydrogenase appeared specific to the WL-SF while angiotensin-converting enzyme, γ-enolase, coagulation factor IX, fibrinogen α-chain, hemoglobin subunit α-D, lysozyme C, phosphoglycerate kinase, Src-substrate protein p85, tubulins and thioredoxin were AIL-specific. The RJF-SF contained fewer immune system process proteins and lower amounts of the anti-inflammatory/immunomodulatory TGF-β2 compared to WL and AIL, which had low levels- or lacked pro-inflammatory CXCL10 compared to RJF. The seminal fluid proteome differs between ancestor and modern chicken, with a clear enrichment of proteins and peptides related to immune-modulation for sperm survival in the female and fertility.

The ubiquitous hyaluronan: Functionally implicated in the oviduct?

Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleukin-10, pertaining maternal immune tolerance of these foreign cells.