Leishmania braziliensis amastigotes stimulate production of IL-1ß, IL-6, IL-10 and TGF-ß by peripheral blood mononuclear cells from nonendemic area healthy residents.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1ß, IL-6, TGF-ß and IL-23 production, whereas IL-10 and TGF-ß are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1ß, IL-6 and TGF-ß. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

Pain location and widespread pain in youth with orthopaedic conditions: Exploration of the reliability and validity of a body map.

BACKGROUND: Pain location and widespread pain are important but underexamined dimensions of paediatric pain. Body map tools to assess pain location in youth have been used for several decades, but few studies have established reliability and validity of these measures. The purpose of this study was to explore the reliability and validity of a pain body map among youth with orthopaedic conditions before surgery. METHOD: Youth ages 10-17 years completed the body map and other self-reported outcomes at their preoperative clinic visit and at their day of surgery. RESULTS: Most (91.7%) youth had small discrepancy between body map scores at preoperative clinic visit (baseline) and day of surgery (second assessment), and site-to-site agreement ranged from 78% to 98%. Those with back and lower extremity diagnoses had high correspondence between body map sites and diagnostic sites. Body map scores and widespread pain were associated with other dimensions of pain, as well as other patient-reported outcomes. Higher pain intensity and widespread pain predicted greater discrepancy between body map scores. CONCLUSIONS: These results support the use of body map tools in further research examining widespread pain among youth by demonstrating adequate reliability, descriptive validity and associative validity. SIGNIFICANCE: These results contribute to the limited information regarding psychometric properties of paediatric pain body maps, provide novel information about widespread pain among youth undergoing orthopaedic surgeries, and pave the way for improved assessment and treatment of paediatric pain.

[Internet use to obtain health information among patients attending a digestive diseases office]. / Uso médico de Internet entre los pacientes de una consulta general de enfermedades del aparato digestivo.

INTRODUCTION: The use of the Internet to gain health information has increased in Spain. This is changing the way patients access medical information and, in turn, the physician-patient relationship. OBJECTIVES: To analyze the use of the Internet for medical purposes by patients attending a digestive diseases office and to determine the profile of patients seeking this information. MATERIAL AND METHODS: A questionnaire was administered to consecutive patients attending a general digestive diseases office for the first time. RESULTS: Four hundred twenty-three patients completed the questionnaire (56% women, 44% men, mean age 42.63 [15.994] years). A total of 2.4% had only not completed basic education, 14% had completed basic education, 32.8% had completed high school, 50.6% had university degrees, and 0.2% did not answer. More than three-quarters (76.4%) had a computer at home and 72% had internet access. Forty-two percent looked for health information on the internet. Seventeen percent had searched for specific information on their illnesses before consulting and 66% believed the internet is a good resource for obtaining medical information. Seventy percent would be interested in a specific web page on digestive disease topics and 75% would use e-mail to consult with their physicians. The group that most frequently looked for medical information consisted of patients aged 45 years or younger with secondary school or university education. CONCLUSIONS: In our environment, patients with digestive diseases routinely use the Internet to search for medical information. These patients have a high degree of confidence in the information obtained and would be interested in a specific website devoted to diseases of the digestive system. The patients that most frequently searched for health information were aged less than 45 years old and had secondary school education or university degrees.

Evaluation of an rK39-based immunochromatographic test for the diagnosis of visceral leishmaniasis in human saliva.

Visceral leishmaniasis (VL) is a tropical neglected disease endemic in 98 countries and affects more than 58 000 individuals per year. Several serological tests are available for VL diagnosis, including an immunochromatographic (IC) test with the rK39 antigen and finger prick-collected blood, a rapid and low-invasive test. Here, we investigate the possibility to use saliva as a non-invasive source of biological material for the rK39 IC test. Blood samples from 84 patients with suspected VL were screened by the rK39 IC test, and 29 were confirmed as being infected by a positive rK39 IC test and the presence of amastigotes on smears slides or parasite DNA (detected using PCR-RFLP) from bone marrow aspirate. The rK39 IC test using saliva samples was positive for 17 of the 29 confirmed VL cases (58.6%). The amount of Leishmania-specific IgG or total IgG, as evaluated by an immunoenzymatic assay, was higher in the saliva of patients who had rK39 IC test positivity using saliva, whereas the amount of Leishmania-specific IgA or total IgA was similar to the healthy donors. These results suggest that saliva is not an appropriated material for diagnosing VL with this test.

Enhanced preovulatory progesterone levels in clomiphene citrate-induced cycles.

We measured basal body temperature, cervical mucus, follicular diameter, and serum LH, FSH, 17 beta-estradiol, and progesterone daily in 40 spontaneous ovulatory cycles in 27 infertile women and in 40 clomiphene citrate-induced ovulatory cycles in 31 women. The cervical score was significantly lower and the serum FSH, LH, and 17 beta-estradiol levels were significantly higher during the follicular phase in induced compared with spontaneous cycles. Serum progesterone was significantly higher on the 3 days preceding ovulation and the day of ovulation in the induced cycles. These increased preovulatory serum progesterone levels could contribute to the lower cervical mucus score in the induced compared with spontaneous cycle, whereas their effect on the endometrium is still unclear.

Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction.

The 35/50 kDa mucin-like surface glycoprotein (gp35/50) of Trypanosoma cruzi metacyclic trypomastigotes has been implicated in mammalian cell invasion. In this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10D8 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. The Ca2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca2+ concentration ([Ca2+]i), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca2+]i significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10D8 and apparently is not involved in target cell invasion.

Identification of a domain of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 required for attachment and invasion of mammalian cells.

Recombinant proteins and synthetic peptides representing various sequences of gp82, a surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes implicated in mammalian cell invasion, were used in this study aiming at the identification of the domain(s) of this molecule required for interaction with target cells. Invasion of cultured HeLa cells by metacyclic trypomastigotes was inhibited by about 80% in the presence of native gp82 or the corresponding recombinant construct J18. Inhibition by recombinant proteins J18a and J18b, containing respectively the N-terminal and the C-terminal portions of gp82, was on the order of 30% and 65%. As compared to J18b (amino acids 224-516), the truncated gp82 fragments J18b1 (amino acids 303-516) and J18b2 (amino acids 357-516) displayed lower inhibitory effect (approximately 40% and approximately 15%, respectively). Compatible with these observations, we found that the recombinant protein J18b, but not J18a or J18b2, binds to HeLa cells in a dose-dependent and saturable fashion. Experiments with ten overlapping synthetic peptides, representing the gp82 portion spanning amino acids 224-333, showed that peptides 4 (amino acids 254-273) and 8 (amino acids 294-313) have significant inhibitory activity on HeLa cell invasion by metacyclic forms. All these results indicate that the portion of gp82 required for mammalian cell attachment and invasion is located in the central domain of the molecule.

Biodegradable laminar implants for sustained release of recombinant human growth hormone.

Due to its short half-life, renal toxicity and necessity for daily subcutaneous injections, recombinant human growth hormone (rhGH) would best be administered in a controlled release formulation. One approach to this is the use of biodegradable laminar implants based on poly(lactic/glycolic) acid. Two PLGA laminar implant formulations (10% w/w), F(1) and F(2), were designed and statistically compared with a conventional commercial injectable (Norditropin). The following variables were chosen as responses: body weight, organ weights (heart, spleen and thymus), and tibia weights and dimensions. After statistical analysis, the most sensitive responses for detecting differences between formulations were body weight, tibia length and organ weights. Initial in vitro studies of these formulations showed an incomplete gradual release (after 15 days 40% rhGH released from F(1) and 60% from F(2)) and in vivo the best results were obtained with F(1). This formulation showed the best growth curve and the highest values of all the above responses. In conclusion, this type of formulation provides a sustained release of rhGH for at least 15 days and a greater efficacy than the frequent administration of the conventional injectable.

Transrectal ultrasonography in the assessment of rectovaginal endometriosis.

OBJECTIVE: To evaluate the validity of transrectal ultrasonography in the assessment of rectovaginal endometriosis. METHODS: We compared the findings of transrectal ultrasonographic examination performed before surgery with the operative and pathologic findings in 140 women who underwent laparoscopy or laparotomy for suspected endometriosis. The ultrasonographer was asked to investigate whether any deep endometriotic lesions were present in the rectovaginal septum and to define the lateral extension on the basis of involvement of the uterosacral ligaments. In addition, infiltration of the rectal and vaginal walls was evaluated. RESULTS: Thirty-four women had endometriosis infiltrating the rectovaginal septum confirmed by combined operative and pathologic findings. Ultrasonography showed a sensitivity and specificity of 97% and 96%, respectively, in the diagnosis of the presence of rectovaginal endometriosis. The sonographer identified infiltration of the rectal and vaginal walls correctly in all cases in whom it was present, but also reported rectal infiltration in three cases not confirmed by the surgeon and pathologist. The sensitivity and specificity in the diagnosis of uterosacral ligament infiltration were 80% and 97%, respectively. CONCLUSION: If our preliminary results are confirmed by a larger series, transrectal ultrasonography will be considered a valid diagnostic tool in the evaluation of rectovaginal endometriosis.

Ultrasound in the diagnosis of subclasses of unicornuate uterus.

Fourteen infertile women with hysterographic diagnoses of unicornuate uterus underwent pelvic ultrasonography and, subsequently, laparoscopy to evaluate the ability of ultrasound to diagnose the subclasses of this malformation. Seven women underwent laparotomy to remove a rudimentary horn. Ultrasound demonstrated a sensitivity of 85.7% and a specificity of 100% in diagnosing the presence of a rudimentary horn, and a sensitivity of 80% and a specificity of 100% in diagnosing the presence of a cavity in the rudimentary horn. Urography confirmed the supplementary information on the urinary tract provided by ultrasound. Ultrasound appears to have a role in the clinical management of women with unicornuate uterus.

Magnetic resonance evaluation of double uteri.

A total of 18 infertile patients with hysterosalpingographic diagnosis of bifid uterus underwent magnetic resonance imaging (MRI) and subsequent laparoscopy or laparotomy to evaluate the capability of MRI to differentiate among the various classes of the malformation. Magnetic resonance imaging identified both bicornuate uteri correctly, both didelphic uteri, nine of the 12 partial septate uteri, and both complete septate uteri. The method demonstrated a sensitivity of 100% and a specificity of 78.6% in demonstrating cervical prolongation of the spur. Compared with laparoscopy, MRI is less expensive and less invasive, and can also be performed in patients with extensive adhesions. However, it has the disadvantage of not providing information on tubal conditions or on the presence of minimal and mild endometriosis.

Transvaginal ultrasonography versus hysteroscopy in the diagnosis of uterine submucous myomas.

Seventy-one women with symptomatic uterine myomas, hospitalized for hysterectomy, underwent preoperative transvaginal ultrasonography and hysteroscopy to compare their reliability in the diagnosis of submucous myomas. After the operation, the surgical specimen was studied carefully and the results were compared with the preoperative diagnostic findings. Transvaginal ultrasonography had a sensitivity of 100% and specificity of 94%; the predictive value of an abnormal ultrasound scan was 81% and that of a normal one was 100%. The sensitivity of hysteroscopy was 100% and the specificity 96%; the predictive value of an abnormal hysteroscopic finding was 87% and that of a normal result was 100%. Mapping of uterine myomas is more precise with transvaginal ultrasonography than with hysteroscopy, but the former method cannot distinguish between a myoma and an endometrial polyp.

Magnetic resonance imaging in Mayer-Rokitansky-Küster-Hauser syndrome.

Six girls with suspected Rokitansky syndrome underwent magnetic resonance imaging (MRI) and then sonography and laparoscopy to confirm the clinical diagnosis and define the precise characteristics of the malformation. The uterus was missing in all; four had müllerian rudiments in place of the uterus, which were cavitary in two as shown by sonography and in one by MRI. Both ovaries were present in all cases, normally located in four and extrapelvic in two; they were correctly identified by MRI in all cases and by sonography in four. Partially canalized vaginal rudiments were recognized by MRI in two girls and by sonography in one. One patient had unilateral renal agenesis, demonstrated by both MRI and sonography. Magnetic resonance imaging was more precise than laparoscopy and sonography in defining the anatomical characteristics of Rokitansky syndrome, and is less invasive and expensive than laparoscopy.

Reproductive performance of women with unicornuate uterus.

The reproductive history of 19 women with a diagnosis of unicornuate uterus confirmed by laparoscopy or laparotomy is analyzed. The patients were followed for 2 to 10 years. One patient had a cavitary communicating rudimentary horn, four a cavitary noncommunicating rudimentary horn, seven a noncavitary rudimentary horn, and seven no rudimentary horn. Six of the patients presented with primary infertility. The other 13 women had a total of 29 pregnancies, 1 (3.4%) in a rudimentary horn determining rupture; abortions occurred in 17 (58.6%), premature labor in 3 (10.3%), and term births in 8 (27.6%), with a live birth rate of 38%. Of the 11 births, 6 (54.5%) were breech presentations, and 9 (81.8%) were cesarean sections. In five cases the rudimentary horn was removed, with associated salpingooophorectomy in three patients. Cervical cerclage was not performed in any of the patients.

Treatment of adenomyosis-associated menorrhagia with a levonorgestrel-releasing intrauterine device.

OBJECTIVE: To evaluate the efficacy and tolerability of treatment with a levonorgestrel-releasing intrauterine device (IUD) in women affected by adenomyosis-associated menorrhagia. SETTING: Tertiary care center. DESIGN: Prospective, open, noncomparative study. PATIENT(S): Twenty-five women aged 38 to 45 years with recurrent menorrhagia associated with adenomyosis diagnosed at transvaginal ultrasonography participated in this study. INTERVENTION(S): An IUD releasing levonorgestel 20 mcg/day was inserted in each patient within 7 days of the start of menstrual flow. All of the patients were requested to compile a pictorial blood loss assessment chart each month. They underwent clinical and transvaginal ultrasound examinations 3, 6, and 12 months after IUD insertion. MAIN OUTCOME MEASURE(S): Menstrual pattern; serum hemoglobin, ferritin, and iron level changes. RESULT(S): One patient experienced IUD expulsion 2 months after device insertion and another requested removal of the IUD 4 months after insertion because of persistent irregular blood loss. Six months after IUD insertion, amenorrhea was observed in 2 patients and oligomenorrhea in another, spotting occurred occasionally in 7, and 13 had scanty but regular flow. One year of follow-up has been completed by the remaining 23 women: 2 with amenorrhea, 3 with oligomenorrhea, 2 with spotting, and 16 with regular flows. Significant increases in hemoglobin, hematocrit, and serum ferritin have been observed, but the lipid metabolism and clotting variables have remained unchanged. CONCLUSION(S): Our findings indicate that marked and safe relief from adenomyosis-associated menorrhagia can be obtained with the use of a levonorgestrel-releasing IUD.

PIP: The efficacy and tolerability of treatment of adenomyosis-related menorrhagia with a levonorgestrel-releasing IUD were investigated in a prospective study of 25 women recruited from a tertiary care center in Italy. All women reported recurrent menorrhagia of at least 6 months' duration and underwent abdominal and transvaginal ultrasonography, hysteroscopy, and endometrial biopsy before study entry. An IUD releasing 20 mcg/day of levonorgestrel was inserted within 7 days of the onset of menses and follow-up examinations were conducted 3, 6, and 12 months after insertion. 1 woman experienced IUD expulsion 2 months after insertion and another requested removal at 4 months because of persistent irregular blood loss. 6 months after IUD insertion, amenorrhea was observed in 2 women, oligomenorrhea in 1 woman, and occasional spotting in 7 women; the remaining 13 women had scanty but regular periods. At 12 months, 2 women reported amenorrhea, 3 had oligomenorrhea, 2 had spotting, and 16 had regular periods. All menstrual anomalies were well tolerated. IUD-related side effects included headache (24%), breast tenderness (16%), seborrhea or acne (24%), and weight gain (28%). Significant increases in hemoglobin, hematocrit, and serum ferritin were recorded; there were no changes in lipid metabolism or clotting variables. These findings suggest that insertion of a levonorgestrel-releasing IUD represents a viable alternative to hysterectomy in the treatment of adenomyosis. It is speculated that the IUD produces deciduation and subsequent marked hypotrophy of eutopic endometrium.

Ultrasonography in the differential diagnosis of "double" uteri.

A total of 43 infertile patients with hysterosalpingographic diagnosis of bifid uterus underwent sonography and subsequent laparoscopy/hysteroscopy to evaluate the capacity of ultrasound to demonstrate the peritoneal profile of the malformed uteri correctly. Sonographic visualization was adequate in 39 cases (90.7%) and the following were identified correctly: 1 of 2 didelphic uteri, all of 11 bicornuate uteri, all of 4 complete septate uteri, and all of 22 partial septate uteri. The sensitivity of ultrasound in detecting the presence of a perimetrial fundal notch was 92.3% and its specificity 100%. Thus, a precise differential diagnosis of "double" uteri was possible with this technique, which may in some clinical situations provide an alternative to laparoscopy.

Structural and ultrastructural defects in preovulatory endometrium of normo-ovulating infertile women with minimal or mild endometriosis.

To investigate whether a primary endometrial factor is involved in the pathogenesis of infertility in patients with minimal or mild endometriosis, we compared light, scanning, and transmission electron microscopic studies of preovulatory endometria of 15 endometriotic patients with 12 normal controls. All the women were infertile and normo-ovulating by standard criteria. Six morphometric indexes were considered. The scanning electron microscopic analysis revealed heterogeneity of the endometrial surface epithelium in 77% of the patients with endometriosis and in 16% of the controls. Glandular and stromal mitoses, basal vacuolated cells, and the ciliated:nonciliated cell ratio was significantly reduced in the endometriosis group compared with the controls. Further studies are needed to clarify the role of the observed endometrial anomalies in the pathogenesis of infertility associated with endometriosis.

Ultrastructural aspects of endometrium in infertile women with septate uterus.

OBJECTIVES: To evaluate the endometrial surface morphology in patients with septate uterus and primary infertility in an attempt to throw light on the question of whether endometrial anomalies are involved in the pathogenesis of infertility in women with mullerian malformations. DESIGN: Endometrial biopsies were performed in eight women with septate uterus and primary infertility during hysteroscopy scheduled in the preovulatory phase of the cycle (when a follicle > 17 mm was identified by ultrasonography and E2 levels were >200 pg/mL [conversion factor to SI unit, 3.671]). Two samples were obtained from each patient, one from endometrium covering the septum and the other from endometrium lining the lateral wall of the uterus. All specimens were examined by scanning electron microscopy. MAIN OUTCOME MEASURES: The number of glandular ostia, the ciliated:nonciliated cell ratio, and the number of cilia on ciliated cells were analyzed in endometrial specimen from both the covering of the septum and the corresponding uterine lateral wall. RESULTS: In five patients septal endometrium showed the following defective preovulatory changes with respect to endometrium of the lateral uterine wall: a reduced number of glandular ostia, irregular nonciliated cells with rare microvilli, incomplete ciliogenesis on ciliated cells, and decrease in the ciliated:nonciliated cell ratio (1:52 +/- 11 versus 1:21 +/- 8). CONCLUSIONS: Our results indicate a decrease in the sensitivity of endometrium covering the septa of malformed uteri to preovulatory hormonal changes. This could play a role in the pathogenesis of primary infertility in patients with septate uterus.

Transvaginal ultrasonography in the diagnosis of diffuse adenomyosis.

OBJECTIVE: To evaluate the diagnostic capability of transvaginal ultrasonography in detecting diffuse adenomyosis. DESIGN: We compared the preoperative transvaginal ultrasound (US) findings and the pathological findings of the surgical specimen in a series of women who underwent hysterectomy for menorrhagia. PATIENTS: Forty-three women (mean [+/- SD] age of 46 +/- 5) with recurrent menorrhagia and enlarged uterus, without evidence of uterine leiomyomas at abdominal US and of endometrial disease at vabra curettage. SETTING: Tertiary care center, university medical school. MAIN OUTCOME MEASURES: Sensitivity, specificity, predictive positive and negative values of transvaginal US in the diagnosis of diffuse adenomyosis. RESULTS: The sonographer diagnosed adenomyosis in 22 patients, whereas the pathologist found adenomyosis in 20 women, confirming the US findings in 16 cases and making an ex novo diagnosis in 4. The sensitivity of transvaginal US was 80%, the specificity 74%, the predictive value of a normal test 81%, and that of an abnormal test 73%. CONCLUSIONS: Transvaginal US seems to represent a real advance in the preoperative diagnosis of diffuse adenomyosis.

New approach to stability assessment of protein solution formulations by differential scanning calorimetry.

A central composite rotate second order design was used to evaluate chicken egg-white lysozyme (lysozyme) thermal stability at different pH, and lysozyme, sucrose and 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentrations, by means of differential scanning calorimetry (DSC). Four measurements were used to characterize the thermogram: the calorimetric enthalpy (DeltaH(cal)), the temperature at maximum heat flux (T(m)), the ratio of maximum heat flux over thermogram area (C(pT(m))/area), and the ratio of calorimetric enthalpies from the second heating cycle to the first enthalpy (R(DeltaH(cal))). These parameters were interpreted using the three step equilibrium model for protein degradation (irreversible degradation following reversible unfolding). In addition to degradation, increased lysozyme concentration leads to a sizeable decrease in DeltaH(cal) and area ratio, showing how it causes protein aggregation; which in turn promotes protein degradation. DeltaH(cal) and T(m) reach maxima at pH 5, R(DeltaH(cal)) at pH 4.19, while C(pT(m))/area increases linearly with pH, revealing a specific base catalysis of the irreversible degradation step. The role of sucrose concentration in lysozyme stabilization is linked to the stabilization of the unfolded moiety; it neither affects DeltaH(cal) nor C(pT(m))/area, but increases both T(m) and R(DeltaH(cal)). No influence of HPbetaCD on the stability of lyzozyme was observed, probably due to low concentrations employed.