Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Left pneumonectomy and expandable prosthesis placement for pleuropulmonary blastoma in a infant: one year follow-up.

Pneumonectomy is a rare procedure in pediatric age. In contrast to adults, children that undergo this intervention are prone to postoperative mediastinal shift, which leads to bronchial stretching resulting in severe respiratory failure. This postpneumonectomy syndrome can be corrected by inserting a prosthesis in the empty side of the chest. The authors present the case of a 9-month-old infant affected with a pleuropulmonary blastoma who underwent left pneumonectomy and prosthesis placement in the same operation.

Light and scanning electron microscopy of Henneguya arapaima n. sp. (Myxozoa: Myxobolidae) and histology of infected sites in pirarucu (Arapaima gigas: Pisces: Arapaimidae) from the Araguaia River, Brazil.

In this report, we describe Henneguya arapaima n. sp., a parasite of the gill arch and gall bladder of Arapaima gigas (pirarucu) collected in the Araguaia River, in the municipality of Nova Crixás, Goiás State, central Brazil. The plasmodia were white, round or ellipsoidal and measured 200-600 microm. Parasite development was asynchronous and the mature spores were fusiform and had smooth wall. The spores measurements were (range, with means+/-S.D. in parentheses): total length-48.4-53.1 microm (51.6+/-3.4 microm), body length-13.5-15.2 microm (14.2+/-0.8 microm), body width-5.1-6.1 microm (5.7+/-0.5 microm), body thickness-4.7-5.3 microm (4.9+/-0.2 microm) and caudal process length-38.0-41.2 microm (38.3+/-2.9 microm). The polar capsules were elongated and of unequal size, with lengths of 6.3-6.8 microm (6.5+/-0.2) and 6.2-6.6 microm (6.3+/-0.1) for the longest and shortest axes, respectively. Capsule width was 1.4-1.6 microm (1.5+/-0.1). Histological analysis showed that the plasmodia occurred in the tunica adventitia of the gall bladder and were delimited by a thin capsule of connective tissue. In the gill arch, the plasmodia were also surrounded by connective tissue similar to the endomesium of striated skeletal muscle cells. Sixty-five juvenile specimens of A. gigas weighing 1.0-25.0 kg were examined, 17 (26.1%) of which were infected. Of these, 14 (82.3%) had cysts in the gall bladder, two (11.7%) had cysts in the gill arch and only one (5.9%) had cysts in both organs. When the fish were grouped by weight, the prevalence of infection in fish weighing up to 10.0 kg (20.7%) was significantly lower than in fish weighing 10.1-25.0 kg (50%) (G=3.93; d.f.=1; p<0.05).

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil / Isolamento e caracterização de Flavobacterium columnare de quatro espécies de peixes tropicais no Brasil

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish, implicated in skin and gill disease, often causing high mortality. The aim of this study was the isolation and characterization of Flavobacterium columnare in tropical fish in Brazil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) and cascudo (Hypostomus plecostomus) were examined for external lesions showing signs of colunmaris disease such as greyish white spots, especially on the head, dorsal part and caudal fin of the fish. The sampling comprised 50 samples representing four different fish species selected for study. Samples for culture were obtained by skin and kidney scrapes with a sterile cotton swabs of columnaris disease fish and streaked onto Carlson and Pacha (1968) artificial culture medium (broth and solid) which were used for isolation. The strains in the liquid medium were Gram negative, long, filamentous, exhibited flexing movements (gliding motility), contained a large number of long slender bacteria and gathered into ‘columns'. Strains on the agar produced yellow-pale colonies, rather small, flat that had rhizoid edges. A total of four Flavobacterium columnare were isolated: 01 Brycon orbignyanus strain, 01 Piaractus mesopotamicus strain, 01 Colossoma macropomum strain, and 01 Hypostomus plecostomus strain. Biochemical characterization, with its absorption of Congo red dye, production of flexirubin-type pigments, H2S production and reduction of nitrates proved that the isolate could be classified as Flavobacterium columnare.

Flavobacterium columnare é o agente etiológico da columnariose em peixes de água doce, ocasionando enfermidade na pele e nas brânquias, provocando freqüentemente um grande número de mortalidade. O objetivo deste estudo foi o isolamento e a caracterização de Flavobacterium columnare em peixes tropicais no Brasil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) e cascudo (Hypostomus plecostomus) foram examinados externamente com relação a sinais característicos de columnariose, como manchas acinzentadas na cabeça, região dorsal e pedúnculo caudal dos peixes. A amostragem compreendeu a coleta de 50 exemplares de peixes, representando as quatro diferentes espécies escolhidas para este estudo. Amostras para o isolamento foram obtidas através de raspado com swab estéril das lesões e do rim dos peixes clinicamente diagnosticados como acometidos por columnarios e imediatamente semeados em meios de culturas artificiais (líquido e sólido) próprios para o estudo de Flavobacterium segundo Carlson e Pacha (1968). No meio líquido, houve o desenvolvimento de microrganismos que observados em gota pendente apresentaram a forma de bacilos finos, longos, móveis por deslizamento. Através da coloração de Gram, apresentaram morfologia de bacilos finos, Gram negativos, agrupados em colunas. Em meio sólido, as colônias eram pequenas, cinza-amareladas, com borda em forma de raiz. No total, foram obtidos quatro isolamentos: 01 cepa de Brycon orbignyanus; 01 cepa de Piaractus mesopotamicus; 01 cepa de Colossoma macropomum; e 01 cepa de Hypostomus plecostomus. A caracterização bioquímica das amostras, como absorção do vermelho Congo, produção de flexirrubina, produção de H2S e redução do nitrato, sugere que os isolamentos poderiam ser classificados como Flavobacterium columnare.

Ileocaecal valve atresia: our surgical approach.

BACKGROUND: Atresia of ileocaecal valve is the rarest type of intestinal atresia; there are very few reports on it in the literature and all cases described to date were treated with ileocolic resection. METHODS: We present a rare case of ileocaecal valve atresia, the third in the literature, in which a "different" technical approach was employed as an alternative to the usual surgical technique (ileocolic resection). The described case came to our attention in a patient presenting with complicated intestinal perforation in whom we performed an ileal stoma after an unsuccessful conservative approach. Approximately four months later we operated her to close the stoma: during this procedure we discovered the colic valve atresia and treated it by creating a "new ileocaecal valve" and carrying out an appendectomy. After a 7-year follow-up no complications have occurred and the girl enjoys very good health. CONCLUSIONS: We conclude that performing a plasty of Bauhin's valve is a valid alternative to intestinal resection in such cases of atresia even if, according to some authors, the resection of the ileocaecal valve was not associated with increased morbidity and mortality (3).

Prenatal diagnosis of female pseudohermaphroditism associated with bilateral luteoma of pregnancy: case report.

Female pseudohermaphroditism associated with luteoma of pregnancy (LP) is a rare condition characterized by varying degrees of masculinization of a female fetus. We describe a case, diagnosed at 13 weeks gestation. Transvaginal ultrasound at 5 weeks of gestation revealed a normal intrauterine gestational sac and an enlarged maternal right ovary. Re-examination at 13 weeks showed a fetus with male external genitalia. Cytogenetic investigation on amniotic fluid revealed a normal female karyotype 46,XX. Follow-up sonography confirmed the previous assignment of male external genitalia and a second amniocentesis was negative for the SRY gene. High levels of androgens were found in the maternal blood. A diagnosis of female pseudohermaphroditism associated with bilateral LP was made. A healthy girl was born by Caesarean section with complete masculinization of external genitalia (Prader V). Histology confirmed a bilateral LP. To the best of our knowledge this represents the first case of prenatal diagnosis of female pseudohermaphroditism associated with LP and demonstrates the feasibility of diagnosis by sonography from 13 weeks gestation. This is also the first case described of Prader V masculinization associated with LP.

High-frequency oscillatory ventilation versus conventional mechanical ventilation in congenital diaphragmatic hernia.

AIMS: Newborns affected by congenital diaphragmatic hernia (CDH) are high-risk patients: today the mortality is still elevated and is essentially due to severe pulmonary hypoplasia, pulmonary hypertension and the absence of surfactant. High-Frequency Oscillatory Ventilation (HFOV) seems to be a good pre- and postoperative technique in cases of CDH. PATIENTS AND METHODS: We report our experience in the treatment of CDH; since 1987 we have followed 44 patients. We divided them into two different groups in accordance with the ventilation technique used: Group I (1987-1994): 25 patients treated with conventional mechanical ventilation (CMV); Group II (1994-1997): 19 patients treated with HFOV used since the first day of life until clinical stabilisation and also prolonged during surgery and in the postoperative period. RESULTS: 37 patients (84%) underwent surgery with closure of the diaphragmatic defect. We had a very good improvement in survival, rising from 67% (CMV) to 94% (HFOV) of the patients operated on, reaching an overall survival of 56% vs 79%. CONCLUSIONS: The use of HFOV for the treatment of CDH has proved to be a valuable technique for pre-operative stabilisation and for intra- and postoperative respiratory treatment, above all for newborns with CDH.

Rhabdomyoma of a rare type in a child: case report and literature review.

We describe a 9-year-old boy who presented with dyspnea and with a neck mass which was initially described as a thyroid nodule. At the end of the diagnostic-therapeutic research this mass was finally diagnosed as an adult rhabdomyoma originating from the mediastinum. This is an extremely rare tumor which is usually found in the adult population.

Origin of enzootic intranasal tumor in the goat (Capra hircus): a glycohistochemical approach.

Enzootic intranasal tumor (EIT) appears glandular in type and has recently been classified as an adenocarcinoma of low malignancy. The aim of this study was to characterize the secretion of surface glycoconjugates (GCs) in EIT and in normal respiratory and olfactory mucosae of the goat by means of conventional and lectin histochemistry, in order to shed light on the histogenesis of EIT. Morphologic and ultrastructural investigations showed two growth types of EIT: i.e., tubular and papillary patterns. Conventional histochemistry revealed the presence of neutral and carboxylated GCs in the olfactory glands and in the tubular part of EIT, as well neutral and sulphated GCs in the respiratory glands and in the papillary part of EIT, suggesting that the papillary pattern tumor arises from the respiratory glands, whereas the tubular portion of EIT arises from the olfactory glands. Lectin histochemistry gave further information on the expressed GCs.

Effects of bilateral microinjections of ibotenic acid in the thalamic reticular nucleus on delta oscillations and sleep in freely-moving rats.

The thalamic reticular nucleus (NRT) consists of a large pool of GABAergic neurons located on each side on the anterior, lateral, and ventral surfaces of the dorsal thalamus. The NRT is divided up into sectors. The aim of this study was to investigate the effects of bilateral lesions of the NRT on sleep and sleep oscillations. Only the results concerning delta oscillations will be reported here. As a first step we produced stereotaxically placed electrolytic lesions. The rats presented continuous circling behavior with electroencephalographic (EEG) theta and delta activity and subsequent sudden death. To avoid disruption of the bundles of fibers that pass through the NRT to and from the cerebral cortex, we used the excitotoxic ibotenic acid. Given its high toxicity, we concentrated on the rostral pole of the NRT, which is believed to have powerful effects on the synchronization of oscillatory activity during sleep. Immediately after surgery, the rats fell into a deep sleep during which there was an increase in EEG slow-wave activity and no spindles. On postoperative day 2, corresponding to the destruction period, the sleep/wake cycle partially recovered, but NREM sleep was quantitatively diminished and showed abnormalities (increased latency to sleep onset, sleep fragmentation, gradual elimination of the delta rhythm). It is concluded that the rostral pole of the NRT contributes to normal and pathological EEG synchronization and the organization of sleep in rats.

[A case of female pseudohermaphroditism caused by maternal androluteoma]. / Caso di pseudoermafroditismo femminile da androluteoma materno.

Female pseudohermaphroditism is a condition characterized by various degree of external genitalia virilization in a patient with female internal genitalia and karyotype (XX). External genitalia is masculinized congenitally when female fetus is exposed to excess androgenic environment. Fetal metabolic abnormalities, like congenital adrenal hyperplasia, are the most common causes of female pseudohermaphroditism, however there is a low incidence of gestational hyperandrogenism caused by maternal pathology. We report a case of female pseudohermaphroditism secondary to a maternal ovarian luteoma of pregnancy producing androgenic hormones. The newborn presented a severe degree of external genitalia virilization with high urogenital sinus (stage Prader V). Moreover we describe the main steps of diagnostic iter that are necessary both to exclude other causes of virilization and to study all anatomical aspects in view of the surgical correction. The operation consists in two phases of action: an early clitorisvulvoplasty according to Passerini-Glazel and a late vaginal pull-through with anterior saggital transanorectal approach (ASTRA).

[Neonatal cystic fibrosis: report of 2 cases]. / Fibrosi Cistica ad esordio neonatale: descrizione di due casi.

Cystic fibrosis is the most common life-limiting recessive genetic disorder in Caucasian. It is caused by mutations of CFTR gene (cystic fibrosis transmembrane conductance regulator); at present over 500 mutations are known. Cystic fibrosis as a cause of respiratory distress in the neonate is quite rare. In neonatal period the most important clinical manifestations are meconium ileum and much rarely cholestatic jaundice. We present two cases of cystic fibrosis in newborns. In the first one, we point out the strict association between meconium ileum and cystic fibrosis. The patient underwent a surgical treatment for meconium ileum and the diagnosis was rapidly confirmed by genetic analysis and sweat test. The second one had intestinal obstruction from birth caused by meconium ileum associated with ileal atresia; besides, he developed cholestatic jaundice, severe and rapidly progressive respiratory disease. He died at 102 degrees day of age for cardiac failure. The diagnosis of cystic fibrosis, supported by typical clinical features and high level of serum trypsin, unfortunately wasn't confirmed by genetic analysis (lambda F508/neg), in addition, the sweat test wasn't reliable because an inadequate quantity of sweat was collected.

Presence and distribution of neuroendocrine cells in the gastroenteropancreatic endocrine system of fallow deer foetuses.

The gastroenteropancreatic endocrine system was studied in 11- and 17-week-old fallow deer foetuses using an immunocytochemical technique. In the gastrointestinal tract, gastrin-, serotonin-, somatostatin- and cholecystokinin-containing cells were found: their frequency and distribution were also determined. Anti-glucagon and anti-insulin antibodies did not stain any cells along the gut. In the pancreas, somatostatin-, pancreatic polypeptide-, insulin- and glucagon-immunoreactive cells were detected. The different distribution and number of neuroendocrine cells, in the two investigated stages of foetal life, are discussed. Data obtained in this study were compared with those published in a previous study on the gastrointestinal system of the adult fallow deer.

Immunohistochemical localization of endometrial oestrogen and progesterone receptors in the cow.

The presence and distribution of oestrogen and progesterone receptors have been investigated by means of immunohistochemical procedures in the uterus of two groups of cows: the first group underwent superovulatory hormonal treatment while the second group was used as a control group. After the immunohistochemical study no differences regarding the presence and distribution of hormone receptors seemed to be apparent between the two groups, so it was concluded that the topographical distribution and staining intensity of hormone receptors seem to be unaffected by hormonal superovulatory treatment.

Anterior sagittal transanorectal approach to the posterior urethra in the pediatric age group.

PURPOSE: Surgical access to the posterior urethra is often difficult and several surgical solutions have been proposed. We suggest an anterior sagittal transanorectal approach based on splitting the anterior rectal wall only. This alternative technique provides excellent exposure to the retrourethral region, permitting simple and safe surgery. MATERIALS AND METHODS: Between 1994 and 1996 we performed surgery via the anterior sagittal transanorectal approach in 8 patients with a mean age of 9.06 years. Patients included 1 girl with a posttraumatic urethrovaginal fistula, 3 with intersex disorders (2 with mixed gonadal dysgenesis raised as boys and 1 with male dysgenetic pseudohermaphroditism with an enlarged urtricle) and 4 boys (1 with penile agenesis raised as girl, 2 with urethral duplication and 1 with prostatic rhabdomyosarcoma). The patient was placed in a knee-chest position. A midline sagittal incision was made through the anterior anorectal wall only and deepened through the perineal body to expose the posterior urethra and retrovesical space. After the pathological condition was corrected the anterior rectal wall and perineal body were reconstructed. The operation was completed with protective colostomy. In our final patient with prostatic rhabdomyosarcoma the anterior sagittal transanorectal approach was used without colostomy. Anorectal manometry was done 6 months postoperatively. RESULTS: All patients were completely continent of stool and urine. Convalescence was unremarkable in all cases. Postoperative manometry in 7 patients revealed no differences from preoperative measurements. CONCLUSIONS: This procedure should be considered a useful alternative to other techniques for various congenital and acquired pelvic disorders.

Esophageal leiomyomatosis in children: report of a case and review of the literature.

The authors describe a case of a 5-years-old girl affected by diffuse oesophageal leiomyomatosis presenting with progressive dysphagia, cyanosis, recurrent pneumonia and retrosternal pain. She also suffered from occasional constipation due to perineal involvement by the disease and had a past history of recurrent microscopic hematuria, suggesting an association with an Alport-like syndrome. Only 24 cases of esophageal leiomyomatosis could be found in the medical literature in children aged less than 14 years, confirming the rarity of the disease. A clinical analysis of all cases reviewed from the literature is made, stressing the importance of an accurate preoperative diagnosis for the choice of proper surgical treatment to avoid recurrence.

Isolated agenesis of the gallbladder. An intraoperative problem.

Agenesis of the gallbladder and cystic duct is a rare congenital malformation. In 40-70% of cases this anomaly is associated with other gastrointestinal, skeletal, cardiovascular and genitourinary malformations. Lithiasis of the common bile duct is present in 25-50% of cases. In the majority of cases patients are asymptomatic or have symptoms compatible with a biliary disorder. A preoperative diagnosis is extremely difficult and the absence of the gallbladder is often an intraoperative finding. The authors report a case of isolated agenesis of the gallbladder. The relative embryology, development, diagnostic pitfalls, intraoperative behaviour and therapeutic strategies are discussed.

Ultrastructural study on the stomach of Tilapia spp (Teleostei).

An ultrastructural study has been made of gastric mucosa of a teleostean fish, Tilapia spp. The cytological features of the surface mucous cells, mucous neck cells, glandular cells and endocrine cells are described. The surface mucous cells, identified by their superficial localization, are characterized by apical granules. The mucous neck cells are distinguished by the appearance of their mucous granules and their localization between surface mucous cells and glandular cells. The gastric glands contain only one form of cell whose fine structure is similar to cells that secrete hydrochloric acid. Physiological implications of some ultrastructural features are also discussed.