Node Fragments Research Articles

Successful experience with axenic isolation of Leishmania infantum in culture medium enriched with equine blood

The aim of the study was to demonstrate a successful experience in the axenic isolation of Leishmania infantum (L. infantum) promastigotes in culture using base agar media enriched with defibrinated equine blood (SDE). The study used lymph node aspirate, spleen, liver and lymph node fragments from dogs infected with the disease. The culture media used for the isolation of the parasite were biphasic (solid and liquid) and monophasic (liquid). For a biphasic phase, Blood Agar Base (BAB) and Neal Novy Nicolle (NNN) medium were used, both enriched with SDE, which after solidification was added to Brain Heart Infusion (BHI) broth. For the monophasic phase, Medium 199 (M199) supplemented with 10% fetal bovine serum (SFB) was used. The promastigotes were seeded in duplicate, kept in an oven by biochemical oxygen demand at 25º C for 15 days. Microscopic readings were taken daily to monitor the primary growth phase of the parasite. After primary isolation, they were serialized and diluted with a final concentration of 1x106 parasites/mL, for evaluation of the growth curve. The continuous study showed that in tubes containing SDE enriched NNN and BAB biphasic media, primary parasite growth was 72 hours, whereas in tubes supplemented with SFB it was 96 hours. Regarding the growth curve, it was observed that during an exponential phase, as SDE enriched spleen, liver and lymph node fragments obtained greater promastigotes, when used with SFB supplemented ones, being 189 x 106 e 128 x 106 parasites/mL, respectively. There was a significant difference (P < 0.05) in the percentage of the number of promastigotes in a medium enriched with SDE, when compared to the medium with SFB. The primary tubes remained viable throughout the study period, even after replenishing used to maintain the parasite. The traditional technique of isolation of the parasite in culture medium most commonly used is in base agar enriched with rabbit blood; however, operational difficulties in the acquisition of this enricher have driven the search for other sources of protein supplementation for L. infantum cultivation. The study reveals that the use of SDE as an alternative enrichment medium for the primary isolation of L. infantum was feasible to be used in laboratory routine.

Nanofibrillar collagen scaffolds for lymphedema treatment: current applications and future directions

Aim: Biosynthetic scaffolds represent cutting-edge therapeutic efforts for secondary lymphedema. In particular, nanofibrillar collagen scaffolds have shown efficacy in both preclinical and clinical contexts, and there has been growing interest in these scaffolds in recent years. This study systematically reviewed the current literature on nanofibrillar collagen scaffolds for lymphedema treatment to synthesize findings and highlight areas for further research. Methods: This was a systematic scoping review of the literature on nanofibrillar collagen scaffolds for lymphedema treatment. Results: Upon review of the literature, 32 relevant articles were identified, of which seven articles specifically investigating nanofibrillar collagen scaffolds were selected for inclusion. Of these articles, three investigated scaffold placement in small or large animal models, while four were clinical investigations ranging from case reports to retrospective cohort studies. Across all studies, scaffold implantation was associated with significant improvement in lymphedema symptoms compared to untreated controls, especially when used in combination with physiologic microsurgical procedures such as vascularized lymph node transfer. However, even when used alone or in combination with lymph node fragments, subcutaneous placement of these scaffolds improved lymphedema symptoms. Additionally, in a rodent model of lymphedema, scaffold placement at the time of lymph node harvest forestalled the development of lymphedema, highlighting the preventative capacity of these scaffolds as well. Conclusion: Nanofibrillar collagen scaffolds have been demonstrated to effectively treat and/or prevent secondary lymphedema in both preclinical and clinical investigations. Ultimately, these scaffolds represent a promising intersection of tissue engineering and lymphedema therapy, and further clinical investigation is warranted.

High prevalence of subclinical paratuberculosis in buffaloes (Bubalus bubalis) in Maranhão, Brazil.

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.

Intercurrence of Paratuberculosis in Intradermal Tuberculin Test Reactive Cattle

Background: Bovine tuberculosis control programs are based on a standard diagnostic method, the intradermal test with purified protein derivatives, which is used to identify and eliminate diseased animals. Currently none of the tests available allow complete differentiation between infected and uninfected animals. The main limitations of the tests available are related to diagnostic sensitivity and specificity, which results in false positive reactions due to the existence of cross infections, and also false negative, inherent to the state of energy of some animals. The aim of this work was to study the intercurrence of paratuberculosis in tuberculosis reactive cattle by the comparative cervical test.Materials, Methods & Results: Three hundred and thirty four cattle were evaluated using the comparative cervical test (CCT) and serology for tuberculosis (TB) and paratuberculosis (PTB) ELISA IDEXX®. All of the animals testing positive by CCT were euthanized and necropsied. Fragments of lymph node, lung and intestine were collected and analyzed using histopathological techniques, with staining by Hematoxylin-Eosin (HE). Samples of lung and lymph nodes (retropharyngeal, submandibular, cervical and mediastinal) of the animals testing positive by CCT were evaluated using qPRC for M. bovis, and intestinal and mesenteric lymph nodes using PCR for PTB. Of the 334 cattle evaluated using the comparative cervical test, 16 were considered positive. No lesions suggestive of tuberculosis were found in the macroscopic inspection of the carcasses. The most evident anatomical and pathological finding was a thickening of intestinal mucosa, found in 12 of the 16 cattle submitted to necropsy. No microscopic lesions suggestive of TB were identified nor was the presence of M. bovis detected by qPCR. The main histopathological findings were observed in the small intestine and mesenteric lymph nodes and identified as enteritis, lymphangitis, lymphangiectasia and granulomatous lymphadenitis. In the intestine the changes are characterized by dilated and inflamed lymphatic vessels and intense inflammatory infiltrate on the mucosa and submucosa. Of the 334 serum samples evaluated, the M. bovis ELISA Antibody Test (IDEXX®) identified 17 positive animals. All the cattle considered positive by M. bovis ELISA were considered negative by CCT. In the samples from nine animals (9/16), DNA from M. avium subsp. paratuberculosis (MAP) was identified and in twelve carcasses (12/16) lesions characteristic of PTB were found, which were subsequently confirmed by histopathological techniques. In another nine animals of the herd anti-MAP antibodies were detected. None of those that tested positive by PTB ELISA were reactive by CCT.Discussion: Animals considered positive by TB ELISA that were not positive in the intradermal test does not mischaracterize the clinical picture of the disease. Considering the inverse relationship between cell-mediated and humoral responses to M. bovis, the intradermal test and the serological tests are designed to measure different immunological responses, which develop during different stages of infection. The progress of the cellular immunological response to humoral immunity occurs in the most advanced stages of tuberculosis. Of the 16 cattle considered positive by CCT, 12 animals presented macroscopic and histological lesions suggestive of PTB and DNA from MAP was detected in nine. Although it is the official test for the control of TB in different countries, the intradermal test with PPD has presented limitations, primarily related to specificity. M. avium subsp. Paratuberculosis is considered the main cause of false positive reactions in the intradermal test. The PPD bacterial extract is a complex mixture of proteins, lipids, sugars and nucleic acids, and many of these components are also shared by numerous species of mycobacteria (tuberculous or not).

Bovine tuberculosis: diagnosis in dairy cattle through the association of analyzes

ABSTRACT: Tuberculosis is a chronic anthropozoonosis of worldwide occurrence, caused by the bacterium Mycobacterium tuberculosis and its variants. In Brazil, the National Program for the Control and Eradication of Brucellosis and Tuberculosis in cattle, is responsible for diagnosing and the correctly allocate positive animals, but there is still a lack of definitive diagnosis of the disease. This study described the use of five diagnostic tools that can be used, preferably together, for the confirmation of suspected cases. These tools included the clinical examination comparative cervical tuberculin test, macroscopic findings during the slaughtering and histopathology of the damaged tissues followed by histochemistry. We evaluated a total of 211 dairy cattle, where 15.1% (32/211) had classic clinical signs of bovine tuberculosis, 74 (35%) showed reactivity in the comparative cervical tuberculin test. Of the total number of animals, 141 (66.8%) were referred for sanitary slaughter due to legal and control issues in the outbreaks of the disease. In the follow-up of slaughtering and inspection of viscera and carcasses, 74 (52.5%) had macroscopic lesions compatible with bovine tuberculosis, while 67 (47.5%) showed no visible changes. During the inspection, fragments of lymph nodes and liver and lung parenchyma were collected from five cattle with macroscopic lesions and five with no lesions. The histopathological analysis showed numerous areas of caseous necrosis with or without central calcification and granulomatous inflammatory infiltrate. In the special staining of Ziehl-Neelsen, numerous acid-fast bacilli were evidenced in all cases.

Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies

High-grade B-cell lymphomas with MYC and BCL2 or BCL6 rearrangements are highly aggressive B-cell lymphomas called double-hit lymphomas (HGBL-DH). They are particularly refractory to standard treatments and carry a poor prognosis. Fragments of resected tumoral lymph nodes from two HGBL-DH patients were put in culture. Continuously proliferating cells were characterized and compared with the original tumors. In both cases, the proliferating cells and the tumor displayed MYC and BCL2 rearrangements. Both cell lines (called LB5848-LYMP and LB5871-LYMP) presented a high proliferation rate and were maintained in culture for more than one year. Upon injection in immunodeficient mice, LB5848-LYMP gave rise to lymphoid tumors. In vitro treatment of these cell lines with a BCL2-inhibitory drug (ABT-199) selectively stopped their proliferation. These new cell lines represent valuable tools for studying HGBL-DH and for the in vitro testing of candidate therapies targeting HGBL-DH. LB5848-LYMP is also suitable for similar experiments in vivo.

Linfossarcoma de células B multicêntrico em uma ovelha

Background : : : Lymphosarcoma is a malignant tumor from tumoral cells similar to lymphocytes and its spontaneous development in small ruminants is unusual in Brazil. In cattle, the lymphosarcomas are often associated with infection by bovine leukemia virus (BLV), although sheep are considered as a very susceptible specie to BLV. Spontaneously lymphosarcoma is rare and in most cases it is observed in apparently healthy animals in slaughterhouses. There is no report of this tumor in sheep in southern Brazil. We describe a case of spontaneous multicentric B-cells lymphosarcoma in an ewe. Case : In June 2007, a 4-years-old mixed breed ewe was necropsied after clinical signs as anorexia, weight loss and death in few weeks. This ewe was purchased in September 2006 with another 44 sheep, most females of the same age. Dairy cattle were also raised together with sheep at the same farm. Fragments of liver, lung, spleen, kidneys, brain and parotid lymph node were sent to the Veterinary Pathology Laboratory - Universidade Federal de Santa Maria, Brazil, for histopathological evaluation. Multiply white to yellow nodules, with 0.5 to 2 cm of diameter, were observed in the liver, kidneys, and the parotid lymph node. The nodules were soft and some coalesced and infiltrating the hepatic parenchyma. The kidneys nodules were similar to those of the liver but prominent in the capsule. They were observed infiltrating more intensely the cortical region. The parotid lymph node was 6 x 4 x 3.5 cm size, whitish, multilobulated and the cut surface was totally obliterated by the tumor mass. Histologically, the neoplasm showed intense proliferation of neoplastic lymphoid cells arranged in sheets and a poor fibrovascular stroma, with round small and hyperchromatic nuclei, and scarce eosinophilic cytoplasm compressing the adjacent parenchyma. Immunohistochemistrily, neoplastic cells were strongly positive for anti-CD79a antibody and negative for anti-CD3 antibody, indicating neoplastic cells derived from B lymphocytes. Discussion : The diagnostic of multicentric B-cell lymphosarcoma was made based on gross, histological, and immunohistochemical findings. Spontaneous development of this tumor is uncommon in sheep in Brazil. The multicentric presentation seen in this case is the most common form of lymphosarcoma in sheep, although the thymic and cutaneous forms also occur. Sheep affected by multicentric lymphosarcomas may have symmetrical lymphadenopathy, not observed in this case, and involvement of various organs including liver, kidney, spleen, intestine, skeletal muscle and heart. However, under experimental conditions, these tumors are easily induced after inoculation of bovine retrovirus (BLV) in sheep, by its high sensitivity. Possibly, the spontaneous transmission of BLV from cattle developing the disease in sheep may occur in Brazil as well as in other countries. Epidemiological factors of the disease are not established yet. Despite this, some correlation must exist for the low occurrence of this disease in sheep in properties of southern Brazil, where cattle and sheep are commonly raised together, and the frequency of lymphosarcoma in sheep is low. In this case, the association of BLV with development of the neoplasm could not be confirmed. This is the first report of multicentric B-cell lymphosarcoma in sheep diagnosed in southern Brazil.

Epstein-Barr virus-positive follicular lymphoma.

A 66-year-old woman presented with left axillary lymphadenopathy. Excisional biopsy was performed. Histological sections showed fragments of an enlarged lymph node with architecture effaced by a vaguely nodular lymphoid neoplasm. The nodules contained predominantly large atypical cells with vesicular chromatin, multiple small nucleoli and variable amounts of cytoplasm, together with a few scattered small cleaved cells (top left). Mitotic figures and apoptotic bodies were variably noted within the nodules. No definite areas of diffuse involvement were noted. The neoplastic cells were positive for CD20, BCL6 (top right), CD10 (focal), CD30 (bottom left), BCL2 and MUM1(focal). In situ hybridisation studies for Epstein–Barr virus (EBV)-encoded small RNAs (EBER) were also performed, showing clusters of positive cells throughout the follicles (bottom right). Lambda immunoglobulin light chain restricted aberrant B cells expressing CD20, CD19 and CD10 (partial/dim) were also detected by flow cytometric analysis. Overall findings were indicative of follicular lymphoma grade 3A. Epstein–Barr virus-positive follicular lymphoma is rare with a prevalence of 2·6%, as reported in the largest case series (Mackrides et al, 2017). Although the majority of cases show EBER-positivity in >75% of tumour cells, low percentage (~5–10%) of EBER-positivity has also been observed. EBV-positivity has been associated with a high grade follicular lymphoma, rapid progression of disease and CD30 expression.

Effect of cryopreservation on lymph node fragment regeneration after autologous transplantation in the minipig model.

Introduction:Lymphoedema is a worldwide pandemic causing swelling of tissues due to dysfunctional transport of lymph fluid. Present management concepts are based in conservative palliation of symptoms through manual lymphatic drainage, use of compression garments, manual lymph drainage, exercise, and skin care. Nevertheless, some curative options as autologous lymph node transplantation were shown to reduce lymphoedema in selected cases. Lately, some concern has arisen due to reports of donor site morbidity. A possible solution could be the development of artificial lymph node scaffolds as niches of lymphatic regeneration. Engineering these scaffolds has included cryopreservation of lymph node stroma. However, the effects of cryopreservation on the regeneration capacities of these organs were unknown.Materials and methods:Here, we used the minipig animal model to assess lymphatic regeneration processes after cryopreservation of autologous lymph nodes. Superficial inguinal lymph nodes were excised and conserved at −80°C for 1 month. Thereafter, lymph node fragments were transplanted in the subcutaneous tissue.Results:Regeneration of the lymph nodes was assessed five months after transplantation. We show that lymph node fragment regeneration takes place in spite of former cryopreservation. Transplanted fragments presented typical histological appearance. Their draining capacity was documented by macroscopic transport of Berlin Blue dye as well as through SPECT-CT hybrid imaging.Discussion:In conclusion, our results suggest that processes of cryopreservation can be used in the creation of artificial lymph node scaffolds without major impairment of lymph node fragments regeneration.

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histological grade of 97 carcinoma samples was attributed, except in situ carcinoma, 37 (38.14%) of the neoplasms were grade I, 50 (51.55%) grade II and only 10 (10.31%) tumors were classified as grade III. Forty bitches were submitted to clinical staging (TNM) and 42.50% of the bitches received staging in grade I and, 25% of the bitches staged in grade IV and V, with metastases. The HER2 expression, 13/99 samples (13.13%) received score +2, 19/99 (19.19%) score +1 and absence of marking (score 0) was identified in 67 samples (67.80 %). Immunostaining in hyperplastic or normal epithelial cells was evidenced, often in association with weak or moderate cytoplasmic labeling. Of the samples expressing +2 score for HER2 (n = 13), eight samples (17.39%) were complex carcinoma and five (9.80%) simple carcinomas. There was no relationship between HER2 immunostaining with age, tumor size, TNM, histological type, histological gradation, lymph node metastasis and distance. Animals with lymph node metastasis, as well as those diagnosed with distant metastasis, did not present HER2 expression in the tumors.Discussion: The simple carcinoma seems to be the most frequent type histological diagnosed in canine mammary carcinomas, followed by carcinoma in mixed tumor and complex carcinoma. Tubulopapillary carcinomas are more invasive in the female dogs as well as in the woman. Carcinomas grade I and II are more frequent and present a better prognosis for the dog. However, bitches with grade III carcinoma survived for a shorter time when compared to dogs with grade I or II tumors. A factor that may have contributed to the lower number of bitches at worst prognostic stage (EC IV and V) is the current owners’ awareness that they have sought veterinary help earlier, as soon as they detect small nodules in mammary gland. Overexpression of HER2 in women breast cancer is diagnosed in 20-30% of cases, whereas in bitches, this expression is variable. Also the different percentages of canine HER2 immunostaining are due to the lack of standardization for the analysis of the immunostaining, the immunohistochemical techniques employed and the non-specificity of the HER2 antibody. In canine mammary carcinomas the HER2 expression in low and this immunostaining is not related to other established prognostic factors. This study reinforces the hypothesis put forward by other authors that in the bitch the expression of HER2 may not be related to malignancy and tumor progression.

Prognostic Value of Occult Isolated Tumour Cells within Regional Lymph Nodes of Dogs with Malignant Mammary Tumours

Canine mammary tumours (CMTs) are the most common type of neoplasm in bitches. As in women, the presence of metastasis in regional lymph nodes is an important prognostic factor in bitches with mammary carcinomas, but the clinical significance of occult isolated tumour cells (ITCs) within lymph nodes is still undefined in this species. The effectiveness of immunohistochemistry (IHC) in identifying occult ITCs and micrometastasis (MIC) was compared with that of the conventional haematoxylin and eosin staining technique. The relationship between tumour size, histological type, histological grade and the presence of metastasis was evaluated. The overall survival (OS) of female dogs with occult mammary carcinomas and ITCs within lymph nodes was analysed. Fragments of mammary carcinoma and regional lymph nodes of 59 female dogs were also evaluated. Histological sections of mammary carcinoma and lymph node samples were studied for tumour diagnosis and lymph node samples were tested by IHC using a pan-cytokeratin antibody. It was found that 35.2% of occult ITCs and 2.8% of hidden MIC were detected when IHC was used. There was a good correlation between the size of the tumour and metastasis to the lymph nodes (P=0.77). ITCs were observed more frequently in the medullary region (60.7%) and metastases in the cortical region (44.4%). There was no significant difference in the OS between female dogs with occult ITCs and lymph nodes without ITCs. IHC can detect occult tumour cells in lymph nodes that are negative by histopathological examination. Female dogs with nodal ITCs do not have lower survival.

Himatanthus drasticus (Apocynaceae) latex reduces oxidative stress and modulates CD4+, CD8+, FoxP3+ and HSP-60+ expressions in Sarcoma 180-bearing mice

Ethnopharmacological relevanceIn Brazil, latex of Himatanthus drasticus is used to treat inflammation, wound healing and cancer. The present study evaluated the antitumoral potential of H. drasticus latex (HdCL) in Sarcoma 180-bearing mice (S180). Materials and methodsHdCL was obtained in Crato-CE, Brazil. Qualitative phytochemicals assays, nuclear magnetic resonance (NMR) and microbiological analyzes were performed. Swiss mice were divided into six groups, according to tumor forms: 1) ascitic model, GI (Control; 0.9% saline), GII (S180asc) and GIII (S180asc/HdCL/14 days); 2) solid model, GIV (Control; 0.9% saline), GV (S180sol) and GVI (S180sol/HdCL/10 days). HdCL and 0.9% saline were administered at 0.2 mL, SID, by gavage, for 10 or 14 days. For ascitic model, 0.5 mL of S180 suspension (4×106 cells/mL) was inoculated intraperitoneally and for solid model, cells were inoculated subcutaneously (25 µL) on the right hind paw of mice. Blood samples were collected for hematological and oxidative stress evaluation. Thickness, volume and weight of paws were measured in solid model. After euthanasia, spleen, liver and kidney were collected in order to assess the relative organ weight. Tissue fragments of paws and popliteal lymph nodes (PLN) were analyzed by H&E and CD4+, CD8+, HSP-60+ and Foxp3+ immunohistochemistry. ResultsHdCL presented milky aspect and pinkish supernatant. Phenols, flavonols, flavanones, free steroids and cinnamoyl derivatives of lupeol, α-amyrin and β-amyrin were detected at the phytochemistry analysis. HdCL did not alter the relative weight of organs, hematological parameters and volume of ascitic fluid recovered. In solid model, HdCL reduced (P < 0.05) paw volume, but did not altered thickness, paw weight and histological parameters. S180sol induced necrosis, metastasis and destruction of bone, cartilage and muscles. Bleeding, vessel congestion and oncocytes were observed in PLN. In paw, HdCL did not alter FoxP3+ and HSP-60+ expressions but reduced the CD4+ and CD8+ expressions, while at PLN, HdCL reduced the expressions of all markers. HdCL decreased (P < 0.05) serum levels of malondialdehyde in ascitic model. ConclusionsTreatment with HdCL reduced oxidative damage and modulated the expressions of CD4+, CD8+, FoxP3+and HSP-60+ in S180 solid tumor model, which can be associated to the presence of triterpenes, such as α-amyrin, β-amyrin and lupeol cinnamate. Present data emphasizes the importance of immune system in cancer and highlights the evaluation of the pharmacological properties of plants used by population as phytoterapics.

Abstract 11422: Aligned Nanofibrillar Collagen Scaffold Guide Therapeutic Lymphangiogenic Repair in Secondary Lymphedema

Background: As a mechanical adjunct to therapeutic lymphatic repair in acquired lymphedema, we have investigated the applicability of guided lymphatic regeneration, through the introduction of a biological scaffold consisting of aligned collagen fibrils. These fibrils provide for cell attachment, alignment, and migration. Methods: We refined a large animal model using minipigs (n=16). We resected the inguinal and popliteal lymphatic system and irradiated the groin area to reproduce the conditions encountered in human cancer-related lymphedema. We assessed the status of lymphedema by detecting interstitial fluid accumulation through bioimpedance, the number of major lymphatic collectors by contrast-enhanced CT, and presence of lymphatic dermal backflow by contrast-enhanced MRI. Results: Three months after lymphatic resection and irradiation, animal subjects were randomized to: (1) scaffolds only, (2) scaffolds impregnated with VEGF-C, and (3) VEGF-C-scaffolds with additional transplantation of autologous lymph node fragments. The control group (4) did not receive any treatment. Seven animals out of 16 developed chronic lymphedema by the pre-defined bioimpedance threshold. The number of lymphatic collectors, as determined by CT, correlated with bioimpedance data. In group 1, there was slight improvement of bioimpedance ratios. In groups 2 and 4 (control), lymphedema persisted. In group 3 lymphedema was reversed in all subjects at post-Rx time-point 6 months. Macroscopic analysis of collectors in the implantation area after intradermal injection of blue dye showed newly developed lymphatic collectors aligned in the direction of the implanted collagen scaffolds. Conclusions: In this porcine acquired lymphedema model, implantation of aligned nanofibrillar collagen scaffolds facilitates the ingrowth of lymphatics across the treatment field. Moreover, simultaneous transplantation of autologous lymph node fragments improves lymphatic regeneration. The biocompatibility of these surgical approaches suggests their potential as an effective treatment option for the human disease.

VEGF-C improves regeneration and lymphatic reconnection of transplanted autologous lymph node fragments: An animal model for secondary lymphedema treatment.

Secondary lymphedema occurs after for example breast cancer surgery and radiation in 20–50% of the patients. Due to the poor outcomes of surgical treatments in the past, the therapy often remains symptomatic. However, avascular transplantation of autologous lymph node fragments (LN-Tx) combined with postoperative injections of vascular endothelial growth factor-C (VEGF-C) emerges as a potential surgical therapy. In this study, adult rats underwent LN-Tx to investigate the following parameters of VEGF-C application: time point, location and dosage. Furthermore, the influences of VEGF-C on lymphatic reconnection and transplant regeneration were analyzed. The reconnection was investigated using intradermally injected blue dye and the regeneration was evaluated histologically using hematoxylin-eosin (H&E) staining and immunohistochemistry. The higher dosage enhanced the reconnection rates significantly and showed a statistical tendency of improving regeneration. An application on early postoperative days and the injection into the medial thigh improved the reconnection significantly. However, these variables did not affect the regeneration statistically. This study confirms that LN-Tx combined with lymphatic growth factor VEGF-C is a possible approach in the therapy of secondary lymphedema and shows the important role of VEGF-C application parameters.

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody levels (p=0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas.

Disseminated Mycobacterium tuberculosis Infection in a Dog

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs.

Improved Regeneration of Autologous Transplanted Lymph Node Fragments by VEGF‐C Treatment

Secondary lymphedema is a common complication after removal of lymph nodes in combination with radiation therapy in the treatment of breast cancer, cervical cancer, and melanomas. Only symptomatic therapies are available at the moment, and lymphedema is for most patients a lifelong condition involving psychological and physical disabilities. Animal models exist to study the pathophysiology of lymphedema but not to study surgical treatments. The aim of this study was to show that regeneration of autologous transplanted lymph node fragments is possible in rats that were irradiated previously locally in the groin and to examine the effects of vascular endothelial growth factor (VEGF)-C injections on the rate of regeneration of transplanted lymph nodes. In all of the animals, inguinal and popliteal lymph nodes and adjacent lymphatic vessels were unilaterally removed and the inguinal region irradiated by a single dose of 15 Gy. Afterward, lymph node fragments were transplanted subcutaneously in the irradiated region. Half of the animals were treated by local VEGF-C injections after transplantation. Four weeks after transplantation, drainage of the leg was tested by injection of blue dye, and the transplanted fragments were removed and examined immunohistologically. We could show that regeneration of autologous transplanted lymph node fragments is possible in areas treated with radiotherapy in the rat. We also documented that transplants can achieve a connection to the lymphatic collectors of the leg. The results suggest that the outcome of regeneration can be improved by injection of VEGF-C in the transplantation area. Thus, lymph node fragment regeneration may be relevant for lymphedema prevention and therapy.

Análise histomorfométrica da matriz extracelular do linfonodo poplíteo de cães naturalmente infectados por Leishmania (L.) chagasi

Nas Américas, a leishmaniose visceral canina é causada por Leishmania (Leishmania) chagasi, um protozoário intracelular obrigatório do sistema fagocítico mononuclear; as principais alterações histológicas associadas a essa doença ocorrem nos em órgãos linfóides. Apesar de o cão ser considerado o principal mantenedor e disseminador da leishmaniose no ambiente urbano, são escassos estudos dos aspectos histopatológicos e histomorfométricos, em cães naturalmente infectados com L. chagasi, que investiguem a interação entre o parasito e a matriz extracelular. Este estudo visou caracterizar e quantificar as alterações dos componentes celulares e da matriz extracelular (colágenos I e III) do linfonodo poplíteo de 22 cães com infecção natural por L. chagasi detectada através da reação de imunofluorescência indireta (RIF) e compará-las com as alterações encontradas no linfonodo poplíteo de 10 cães não-infectados, negativos na RIF e clinicamente saudáveis. Fragmentos dos linfonodos foram seccionados longitudinalmente, processados rotineiramente para exame histológico e corados por hematoxilina-eosina. Cortes adicionais do mesmo linfonodo incluídos em glicol metacrilato foram corados pelo azul de toluidina para histomorfometria. Linfonodos de cães infectados apresentaram linfadenopatia generalizada, aumento do tamanho e do número dos folículos linfóides, hipertrofia da cápsula e hiperplasia linfóide significativa. Nos linfonodos de cães do grupo infectado, a análise quantitativa de fibras colágenas mostrou significativo predomínio do colágeno I sobre o colágeno III. Esses resultados demonstram que cães infectados por L. chagasi apresentam degradação dos constituintes da matriz extracelular e conseqüente destruição do arcabouço linfóide, alterando a morfologia do órgão.

Effects of autotransplanted lymph node fragments on the lymphatic system in the pig model

Secondary lymphedema often develops after removal of lymph nodes in combination with radiation therapy, in particular in patients with breast cancer, inguinal cancer, cervical cancer and melanoma. No convincing treatment for the prevention and therapy of acquired lymphedema exists so far, therefore we wanted to show the reintegration of transplanted avascular lymph node fragments in the lymphatic system and positive effects of the transplanted fragments on the restoration of the lymphatic flow in this study. A total of 26 minipigs underwent lymphadenectomy of both groins. A minimum of one lymph node was retransplanted. The lymph nodes were cut into small pieces and retransplanted in the left groin (n = 17) or in both groins (n = 9). Different retransplantation techniques were investigated, transplantation of large versus small fragments, with and without capsule. The lymph flow was evaluated 5 and 8 months after surgery, using SPECT/CT and Berlin Blue. The results were confirmed by dissection. The lymph node transplants were assessed histologically. In contrast to the lymph flow in the transplanted groin, the lymph flow in the non-transplanted groin was often malfunctioning. Large lymph node fragments were found reintegrated in the lymphatic system more often than small slices of lymph node fragments. About 5 months after surgery impairment of lymph flow was seen especially after retransplantation of small slices of lymph node fragments. In seven out of eight minipigs a dermal backflow developed in the non-transplanted groin, 8 months after surgery. Only one minipig of these groups developed dermal backflow in both groins. All lymph node fragments showed an organized structure histologically. Autologous lymph node transplantation has positive effects on the regeneration of lymph vessels and restoration of lymph flow after lymphadenectomy.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x10(5) "P" strain oocysts, GII, 5.0x10(6) "RH" strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.