Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Identifying possible contingencies during descriptive analyses of severe behavior disorders.

Contingencies of reinforcement involve, in part, relations between behavior and subsequent environmental events. In this study we observed 11 individuals with developmental disabilities and severe behavior problems while they interacted with their primary care providers in simulated environments (hospital therapy rooms). We compared the probability of obtaining attention, escape from instructional demands, or access to materials following instances of problem behavior with the background probability of those events. However, the focus of our analysis was the evaluation of comparative probabilities ("contingency values") in the context of relevant establishing operations such as diverted attention, instructional demands, and restricted access to materials. Results showed that the method was useful in identifying relations between behavior and subsequent environmental events. Implications for linking descriptive and functional analyses are discussed, and difficulties in identifying naturally occurring contingencies are considered.

One thousand video-urodynamic studies in children with non-neurogenic bladder sphincter dysfunction.

OBJECTIVE: To ascertain the aetiology and epidemiology of non-neurogenic bladder sphincter dysfunction (NNBSD) by assessing the results of prospective video-urodynamic studies (VUD) in 1000 children. PATIENTS AND METHODS: During a 4-year study period (January 1995 to December 1998) 1000 children prospectively underwent VUD to further define their NNBSD. After a noninvasive screening assessment consisting of a history, voiding diary, clinical examination, urine analysis, ultrasonography and uroflowmetry, those children who would benefit from further VUD were selected. The selection criteria included a history of urinary tract infection (UTI), a small bladder capacity not responding to training, dysfunctional uroflow, ultrasonographic abnormalities and resistance to therapy. During the study period 3500 children were screened for incontinence problems, including monosymptomatic nocturnal enuresis; 1000 of these were selected for VUD (524 boys and 476 girls). RESULTS: The urodynamic diagnosis was of normal bladder-sphincter function in 62 (6.2%, male : female 44 : 56), urge syndrome in 582 (58%, 58 : 42), dysfunctional voiding in 316 (32%, 49 : 51) and 'lazy bladder' in 40 (4%, 20 : 80). Boys diagnosed with a 'lazy bladder' were younger than those with urge syndrome and dysfunctional voiding. Girls with dysfunctional voiding were younger than those with urge syndrome. The incidence of UTI was significantly higher in girls than in boys; boys with NNBSD had no greater risk for UTI and in girls the general risk was 34%. Only in girls with a lazy bladder was there a significantly higher incidence of UTI (53%). Reflux occurred equally in all groups, with an overall incidence of 15%. The incidence of obstipation was significantly higher in girls with a lazy bladder, and overall was 17%. CONCLUSION: These results from a large series provide a new insight into the epidemiology and pathophysiology of NNBSD. The age distribution provides evidence against a dysfunctional voiding sequence. The risk of developing UTI in NNBSD is greater only in girls. In children with a lazy bladder the risk is also significantly higher, indicating that residual urine is a greater risk factor than detrusor instability. Urge syndrome and dysfunctional voiding in girls carry the same risk for developing UTI, indicating that bladder instability is a higher risk factor for UTI than detrusor sphincter discoordination. All dysfunctions carry an equal risk for developing secondary reflux. Children with NNBSD have different primary diseases but all have a common risk of incontinence, UTIs, reflux and obstipation.

A method for analyzing changes in response efficiency.

Although experimental effects typically are evaluated by summarizing levels of responding across time (e.g., calculating the mean levels of problem behavior during 10-min sessions), these data summaries may obscure important mechanisms that may be responsible for changes in responding. A case study is reported to illustrate alternative methods of data analysis when decreasing trends in responding may be due to increases in response efficiency.

Further analysis of idiosyncratic antecedent influences during the assessment and treatment of problem behavior.

Recent research findings suggest that idiosyncratic variables can influence the outcomes of functional analyses (E. G. Carr, Yarbrough, & Langdon, 1997). In the present study, we examined idiosyncratic environment-behavior relations more precisely after identifying stimuli (i.e., a particular toy and social interaction) associated with increased levels of problem behavior. Two children, an 8-year-old boy with moderate mental retardation and a 5-year-old boy with no developmental delays, participated. Results of functional analyses for both children indicated that idiosyncratic antecedent stimuli set the occasion for occurrences of problem behavior (hand biting or hand flapping) and that problem behavior persisted in the absence of social contingencies. Further analyses were conducted to identify specific components of the stimuli that occasioned problem behavior. Treatments based on results of the analyses successfully reduced self-injury and hand flapping.

Variable-time reinforcement schedules in the treatment of socially maintained problem behavior.

Noncontingent reinforcement (NCR) consists of delivering a reinforcer on a time-based schedule, independent of responding. Studies evaluating the effectiveness of NCR as treatment for problem behavior have used fixed-time (FT) schedules of reinforcement. In this study, the efficacy of NCR with variable-time (VT) schedules was evaluated by comparing the effects of VT and FT reinforcement schedules with 2 individuals who engaged in problem behavior maintained by positive reinforcement. Both FT and VT schedules were effective in reducing problem behavior. These findings suggest that VT schedules can be used to treat problem behavior maintained by social consequences.

Effects of reinforcement magnitude on spontaneous recovery.

Extinction of operant behavior has been associated with a number of undesirable effects. One such effect is the temporary reappearance of behavior after responding appears to be completely extinguished, known as spontaneous recovery. In this report, the occurrence of spontaneous recovery and its attenuation with large amounts of reinforcement were examined during the treatment of disruption.

Within-session patterns of responding during functional analyses: the role of establishing operations in clarifying behavioral function.

Functional analysis procedures have been demonstrated to be effective for identifying the operant mechanisms underlying problem behavior. However, functional analyses sometimes yield results that are undifferentiated (i.e., show similar levels of responding across test conditions). Within-session (i.e., minute-by-minute) analyses of response patterns during undifferentiated functional analyses have proven useful in clarifying behavioral function. This study extends previous research by examining within-session changes in responding associated with variations in relevant establishing operations. Levels of problem behavior during the presentation and removal of reinforcement were compared when responding occurred in test conditions associated with sources of social reinforcement (i.e., access to attention, materials, escape). Results showed that changes in responding associated with changes in relevant establishing operations could be examined to clarify behavioral function.

Fixed-time schedules attenuate extinction-induced phenomena in the treatment of severe aberrant behavior.

We compared the effects of extinction (EXT) and fixed-time (FT) schedules as treatment for severe problem behavior displayed by 3 individuals with developmental disabilities. First, functional analyses identified the reinforcers maintaining aberrant behavior for all 3 individuals. Next, EXT and FT schedules were compared using a multielement design. During EXT, the reinforcer maintaining problem behavior was withheld. During FT, the reinforcers were presented response independently at preset intervals. Results showed that FT schedules were generally more effective than EXT schedules in reducing aberrant behavior. FT schedules may be used in situations when extinction-induced phenomena are problematic.

Value of transrectal prostatic echography, prostate-specific antigen and rectal examination in the diagnosis of prostate cancer. Relationship with the result of prostatic biopsies.

UNLABELLED: We retrospectively reviewed the files of patients who had undergone a prostatic biopsy between July 1992 and December 1994. A total of 368 biopsies of 340 patients between 36 and 89 years old, could be used. The pathological examination was used to make the diagnosis of prostate cancer. The specificity and sensitivity values of the diagnostic examinations (TRUS, DRE, PSA and PSAD) were calculated. An abnormal TRUS image (hypodens or hyperdens area or irregular prostate) has the best sensitivity: 91.3%. PSA has the best specificity: 57.2%. The specificity and sensitivity of PSA can be improved by using the PSAD: a PSAD limit of 0.15 has a specificity of 66.4% and a sensitivity of 88.9%. The specificity and sensitivity values are also calculated for several combinations of tests. The combination of abnormal DRE or PSAD higher than 0.15 has a sensitivity of 96.2% and a specificity of 35.0%. CONCLUSION: the combination of abnormal DRE or high PSAD has a high sensitivity with a relatively good specificity. We advise to take biopsy if two or more of the examinations are suspect or if one of them is clearly abnormal.

Results of the endonasal surgical closure of nasoseptal perforations.

Eleven patients with nasal septal perforations were surgically treated within a period of seven years. Former septal surgery was the major cause of nasoseptal perforations. The technique most frequently used was a Cottle septoplasty with closure of the perforation by bipedicled flaps and interposition of autogenous cartilage and bone. The authors were inspired by Younger and Blokmanis's model and they used the following parameters to evaluate the results: location, size, symptoms pre- and post-operatively and also rhinoscopia pre- and post-operatively. In nearly all cases the perforation was closed with a decrease of the symptoms.

Results of oral steroid treatment in nasal polyposis.

Twenty-five patients with massive nasal polyposis were treated during four days with 60 mg oral prednisolone (Deltacortril) followed by a progressive reduction of the dose (minus 5 mg per day). Therapeutic efficacy was evaluated by means of symptoms, anterior rhinoscopy, and a CT scan of the sinuses before and at the first visit after the treatment. Seventy-two per cent of the patients showed subjective improvement due to the involution of polyps in the nasal cavity. However, on CT of the paranasal sinuses only 52% showed a clear improvement. In general, the frontal and sphenoid sinuses were more likely to clear up completely than were the ethmoidal and maxillary sinuses. Therapeutic efficacy seemed to be better in the group of ASA-intolerant patients and worse in the allergic group. The "intrinsic" group showed an equal distribution of good results. Also, the presence of eosinophils in polyps played no role in the therapeutic outcome. We found a strong tendency of recurrence within five months after successful oral steroid therapy. On the other hand, pre-operative systemic steroid therapy considerably facilitated the surgical procedure. Keeping in mind the side effects of systemic steroid therapy, this treatment should be mainly reserved for cases in which surgery is planned.

Comparative effects of ketoconazole on rat, dog and human testicular steroidogenesis.

Ketoconazole is an antifungal azole derivative which also inhibits the cytochrome P-450(17)alpha, catalyzing the conversion of progestins into androgens. The effects of ketoconazole on human, dog and rat testosterone biosynthesis were compared using short term incubations of dispersed testicular cells. The results showed that ketoconazole inhibited androgen biosynthesis at lower concentrations in dispersed human testicular cells (IC50: 0.08 mumol/l) than in canine (IC50: 0.1 mumol/l) and rat cells (IC50 greater than or equal to 0.2 mumol/l). Furthermore, they demonstrated that ketoconazole first inhibited the 17,20-lyase activity and then the 17-hydroxylation in rat and dog cells whereas only the 17-hydroxylation was affected in human cells.

Clinical aspects in the surgical treatment of varicocele in subfertile men. I. Comparison of observed and expected pregnancy rates.

Ninety-six couples, consulting for primary (n = 73) or secondary (n = 23) infertility, and thought to be infertile due to a varicocele in the husband associated with sperm abnormalities, were subjected to a follow-up study. Of these, 57 were surgically corrected and 39 were not, depending only on the patient's choice. All women were intensively treated where necessary. From our data we conclude that (1) fertility prognosis is significantly better in couples with secondary subfertility than in couples with primary subfertility, (2) surgical correction of varicocele in cases of secondary subfertility does not increase subsequent pregnancy rates, and (3) surgical correction of varicocele in cases of primary subfertility significantly increases subsequent pregnancy rates, since cumulative observed pregnancy rates are more than double the cumulative theoretically expected treatment-independent pregnancy rates.

Clinical aspects in the surgical treatment of varicocele in subfertile men. II. The role of the epididymal factor.

Ninety-six couples, consulting for primary (n = 73) or secondary (n = 23) infertility, and thought to be infertile due to a varicocele in the husband associated with sperm abnormalities, were subjected to a follow-up study. Of these, 57 were surgically corrected and 39 were not, depending only on the patient's choice. All women were intensively treated where necessary. All patients who were corrected were subjected to scrotal exploration in a search for epididymal anomalies. From our data we conclude that (1) on a statistical basis, comparing observed with expected pregnancy rates, varicocelectomy is useful in primary infertility and unnecessary in secondary infertility and (2) on an individual basis, patients with primary infertility and 'pure' varicoceles have a better prognosis than patients with concomitant epididymal dysfunction.