[Sonographic studies on the significance of follicle size and corpus luteum morphology in dairy cows after insemination]. / Sonografische Verlaufsuntersuchungen zur Bedeutung der Follikelgröße und Gelbkörpermorphologie bei Milchkühen post inseminationem.

OBJECTIVE: In dairy cattle, numerous factors determine the success of an insemination. The aim of the present study was to generate findings concerning the association between the preovulatory follicles, the corpora lutea with or without cavities and pregnancy in dairy cows under field conditions. MATERIAL AND METHODS: Data was obtained from 176 dairy cows scheduled for artificial insemination. The cows were gynecologically examined using sonography at the time of insemination, 24 hours later, on day 9, on day 34 and after day 42 after insemination. Additionally, blood samples were collected from the coccygeal vein at the time of insemination and on day 9 in order to determine blood progesterone level. RESULTS: Depending on the result of the pregnancy test, no difference was detected between the dimensions of the follicles, corpora lutea as well as their cavities and progesterone levels 9 days after insemination in the dairy cows with spontaneous ovulations. In contrast to the corpus luteum without cavity, the surface area of the corpus luteum with cavity remained constant during the study period, while at the same time the cavity decreased in size to a significant degree. In addition, breed differences in corpus luteum sizes were detected on day 34. CONCLUSION: No cause was detected for the formation of cavities in corpora lutea and there was no link to the follicle from which the corpus luteum developed. CLINICAL RELEVANCE: Both the changes in corpus luteum sizes over time and their breed-related differences could have an impact on insemination outcome.

A Micro-Immunotherapy Sequential Medicine MIM-seq Displays Immunomodulatory Effects on Human Macrophages and Anti-Tumor Properties towards In Vitro 2D and 3D Models of Colon Carcinoma and in an In Vivo Subcutaneous Xenograft Colon Carcinoma Model.

In this study, the immunomodulatory effects of a sequential micro-immunotherapy medicine, referred as MIM-seq, were appraised in human primary M1 and M2 macrophages, in which the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-12, IL-23, and tumor necrosis factor (TNF)-alpha, was inhibited. In addition, the potential anti-proliferative effects of MIM-seq on tumor cells was assessed in three models of colorectal cancer (CRC): an in vitro two-dimensions (2D) model of HCT-116 cells, an in vitro tri-dimensional (3D) model of spheroids, and an in vivo model of subcutaneous xenografted mice. In these models, MIM-seq displayed anti-proliferative effects when compared with the vehicle. In vivo, the tumor growth was slightly reduced in MIM-seq-treated animals. Moreover, MIM-seq could slightly reduce the growth of our spheroid models, especially under serum-deprivation. When MIM-seq was combined with two well-known anti-cancerogenic agents, either resveratrol or etoposide, MIM-seq could even further reduce the spheroid's volume, pointing up the need to further assess whether MIM-seq could be beneficial for CRC patients as an adjuvant therapy. Altogether, these data suggest that MIM-seq could have anti-tumor properties against CRC and an immunomodulatory effect towards the mediators of inflammation, whose systemic dysregulation is considered to be a poor prognosis for patients.

Ultra-Low Dose Cytokines in Rheumatoid Arthritis, Three Birds with One Stone as the Rationale of the 2LARTH® Micro-Immunotherapy Treatment.

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are two cytokines involved in the perpetuation of the chronic inflammation state characterizing rheumatoid arthritis (RA). Significant advances in the treatment of this pathology have been made over the past ten years, partially through the development of anti-TNF and anti-IL-1 therapies. However, major side effects still persist and new alternative therapies should be considered. The formulation of the micro-immunotherapy medicine (MIM) 2LARTH® uses ultra-low doses (ULD) of TNF-α, IL-1ß, and IL-2, in association with other immune factors, to gently restore the body's homeostasis. The first part of this review aims at delineating the pivotal roles played by IL-1ß and TNF-α in RA physiopathology, leading to the development of anti-TNF and anti-IL-1 therapeutic agents. In a second part, an emphasis will be made on explaining the rationale of using multiple therapeutic targets, including both IL-1ß and TNF-α in 2LARTH® medicine. Particular attention will be paid to the ULD of those two main pro-inflammatory factors in order to counteract their overexpression through the lens of their molecular implication in RA pathogenesis.

Jawbone Cavitation Expressed RANTES/CCL5: Case Studies Linking Silent Inflammation in the Jawbone with Epistemology of Breast Cancer.

BACKGROUND: The role of signaling pathways as part of the cell-cell communication within cancer progression becomes a crucial area. Chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted), also known as the chemokine C-C motif ligand 5 (CCL5) (R/C), is a protein on which cancer research focus due to its link with aggressive cancer development. OBJECTIVE: Research on fatty-degenerative osteonecrosis in jawbone (FDOJ) shows striking overexpression of R/C in these areas. Here we try to elucidate a potential link between jawbone-derived R/C and breast cancer (BC) and compare these findings by immunohistochemical staining. METHODS: Thirty-nine FDOJ samples extracted from 39 BC patients and samples from 19 healthy control were analyzed for R/C expression using bead-based Luminex® analysis. R/C levels from 5 BC patients were measured in serum before and after FDOJ surgery. Bone density, histology, R/C expression, and immunohistochemistry were analysed in 4 clinical case studies. The R/C staining of two FDOJ BC patients is compared with the immunohistochemical staining of BC cell preparations. RESULTS: A high overexpression of R/C was seen in all FDOJ samples. R/C levels in serum were statistically downregulated after FDOJ surgery (p=0.0241). DISCUSSION: R/C induced "silent inflammation" in BC is widely discussed in scientific papers along with R/C triggering of different signaling pathways, which might be a key point in the development of BC. CONCLUSION: Hypothesis that FDOJ may serve as a trigger of BC progression through R/C overexpression was set by the authors, who thus inspire clinicians to make aware of FDOJ throughout the dental and medical community in BC cases.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BACKGROUND: We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein-2 (BMP2) and BMP4 micro-immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. METHODS: We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1ß. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization-related enzymes (matrix metalloproteinase-1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long-term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. RESULTS: LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re-establish the impaired collagen metabolism induced by IL1ß treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. CONCLUSIONS: LD BMP4 MI treatment shows anti-inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

Potential Role of the Micro-Immunotherapy Medicine 2LALERG in the Treatment of Pollen-Induced Allergic Inflammation.

In this study, we evaluated the efficacy of a micro-immunotherapy medicine (MIM), 2LALERG, in a preclinical model of allergic respiratory disease sensitized with birch pollen extract (BPE). BALB/c mice were immunized with BPE, or saline solution, and were then challenged. Micro-immunotherapy medicine pillules were diluted in water, and 3 doses (0.75; 1.5; 3 mg/mouse) were tested and compared to vehicle control (3 mg/mouse). Treatments and vehicle were orally administered by gavage for 10 days. Micro-immunotherapy medicine (0.75 mg/mouse) reduced the number of total cells as well as the levels of interleukin (IL)-13 in bronchoalveolar lavage fluid (BALF) compared to vehicle control. Eosinophils in BALF tended to be lower compared to vehicle group, and the difference is close to significance. Histological analysis in the lungs confirms a moderate effect of MIM (0.75 mg/mice) on inflammatory infiltration and mucus production. Serum levels of IL-5 in MIM (0.75 mg/mouse)-treated mice were lower compared to vehicle; IL-4 levels tended to be lower too. Total immunoglobulin E (IgE) decreased in serum of MIM (1.5 and 0.75 mg/mouse) groups compared to vehicle control. Micro-immunotherapy medicine exerted the highest effect at the lowest dose tested. Micro-immunotherapy medicine resolved the local and systemic inflammation, even if partially, in a model of pollen-induced, IgE-mediated inflammation.

The Micro-Immunotherapy Medicine 2LARTH® Reduces Inflammation and Symptoms of Rheumatoid Arthritis In Vivo.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which can cause cartilage and bone damages as well as pain and disability. In order to prevent disease progression, reduce pain, and major symptoms of RA, one good strategy consists in targeting proinflammatory cytokines that have the key role in the vicious circle of synovial inflammation and pain. The micro-immunotherapy medicine (MIM) 2LARTH® targets cytokines involved in inflammation. AIM: The aim of the study is to evaluate the effect of the MIM compared to vehicle in an in vivo model of RA, induced in mice after immunization with articular bovine type II collagen. METHODS: Vehicle and MIM were dissolved in pure water (1 capsule in 100 ml) and 100 µl was given by gavage daily for 14 days. To evaluate the severity of arthritis, wrist and ankle thickness was determined, paw edema was measured, and a clinical score from 0 to 4 was established. Furthermore, histological analysis was performed. To evaluate systemic inflammation, circulating levels of IL-1ß and TNF-α were measured by ELISA. RESULTS: Ankle thickness was found to be significantly reduced in MIM-treated mice compared to vehicle-treated mice (P < 0.05) and compared to untreated me (P < 0.05) and compared to untreated me (P < 0.05) and compared to untreated me (ß and TNF-α were measured by ELISA. P < 0.05) and compared to untreated me (. CONCLUSION: The results indicate that the tested medicine reduces inflammation, histological, and clinical signs of RA in a CIA model.

Pro-Inflammatory Cytokines at Ultra-Low Dose Exert Anti-Inflammatory Effect In Vitro: A Possible Mode of Action Involving Sub-Micron Particles?

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are pro-inflammatory cytokines involved in acute and chronic inflammatory diseases. Indeed, immunotherapy blocking these 2 cytokines has been developed. Micro-immunotherapy (MI) also uses ultra-low doses (ULD) of pro-inflammatory cytokines, impregnated on lactose-sucrose pillules, to counteract their overexpression. The study has been conducted with 2 objectives: examine the anti-inflammatory effect in vitro and the capacity of 2 unitary medicines, TNF-α (27 CH) and IL-1ß (27 CH), to reduce the secretion of TNF-α in human primary monocytes and THP-1 cells differentiated with phorbol-12-myristate-13-acetate, after lipopolysaccharide (LPS) exposure; then, investigate the presence of particles possibly containing starting materials using tunable resistive pulse sensing technique. The results show that the unitary medicines, tested at 3 pillules concentrations (5.5, 11 and 22 mM), have reduced the secretion of TNF-α in both models by about 10-20% vs. vehicle control, depending on concentration. In this exploratory study, particles (150-1000 nm) have been detected in MI ULD-impregnated pillules and a hypothesis for MI medicines mode of action has been proposed. Conscious that more evaluations are necessary, authors are cautious in the conclusions because the findings described in the study are still limited, and future investigations may lead to different hypothesis.

Effect of the micro-immunotherapy medicine 2LPARK® on rat primary dopaminergic neurons after 6-OHDA injury: oxidative stress and survival evaluation in an in vitro model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by motor impairments and resulting from progressive degenerative loss of midbrain dopaminergic (DAergic) neurons in the substantia nigra. Although the main cause of the loss of DAergic neurons is still unknown, various etiopathogenic mechanisms are distinguished, including release and accumulation of endogenous excitotoxic mediators along with the production of oxidative free radicals. Several neurotrophic and growth factors are known to increase DAergic neuronal survival and enhance antioxidant mechanisms. In this context, the micro-immunotherapy (MI) approach consists to regulate the immune system in order to protect DAergic neurons and control oxidative stress. PURPOSE: The aim of the present study was to investigate the effect of the MI medicine (MIM), 2LPARK® (Labo'Life), on oxidative stress and on the number of neurons positive for tyrosine hydroxylase (TH), in an in vitro model of PD. METHODS: Rat primary mesencephalic DAergic neurons cultures were pre-treated for 1 hr with the MIM (10 µM and 10 mM), placebo (10 µM and 10 mM) or brain-derived neurotrophic factor (BDNF; 3.3 µM) and then intoxicated with 6-hydroxydopamine (6-OHDA; 20 µM) for 48 hrs. After incubation, cells were incubated 30 mins at 37°C with CellROX green reagent and number of labeled cells were quantified. Then, cells were fixed and incubated with anti-TH antibody and the number of TH+ neurons was evaluated. RESULTS: We showed that, contrary to placebo, MIM was able to reduce oxidative stress and protect DAergic neurons from 6-OHDA-induced cell death. CONCLUSION: Our results demonstrate the in vitro efficacy of MIM on two essential mechanisms of PD and propose the MI approach as a new ally in the regulation of neuroinflammation and in the treatment of this degenerative disease.

2LARTH®, a micro-immunotherapy medicine, exerts anti-inflammatory effects in vitro and reduces TNF-α and IL-1ß secretion.

BACKGROUND: Tumor necrosis factor-α (TNF-α) and IL-1ß are 2 pro-inflammatory cytokines known to be involved in rheumatic diseases. The therapeutic strategy used in micro-immunotherapy (MI) to reduce chronic inflammation and attenuate pain consists in mainly targeting these 2 cytokines. 2LARTH® is a sublingually administered medicine consisting of lactose-saccharose globules impregnated with ethanolic preparations of immune mediators and nucleic acids at ultra-low doses. PURPOSE: The aim of the study is to explore the effect of the MI medicine on TNF-α and IL-1ß secretion in human primary enriched monocytes exposed to lipopolysaccharide (LPS). MATERIALS AND METHODS: Placebo and active globules were diluted in culture medium to test 5 lactose-saccharose globules concentrations (from 1.75 to 22 mM). Freshly isolated enriched monocytes from 6 healthy donors were treated with or without LPS (10 ng/mL), LPS+ placebo, or LPS+ 2LARTH® for 24 hours. IL-1ß, TNF-α, and IL-6 release were evaluated by ELISA. RESULTS: The medicine has significantly decreased the level of IL-1ß secretion compared with placebo at these concentrations: 22 mM (P<0.0001), 11 mM (P=0.0086), 5.5 mM (P= 0.0254), and compared with untreated LPS control at these concentrations: 22 mM, 11 mM (P=0.0008), and 5.5 mM (P=0.002). The effect of active globules on the reduction of TNF-α release is significant compared with placebo at these concentrations: 22 mM (P=0.0018), 11 mM (P=0.0005), 5.5 mM (P=0.0136), and compared with untreated LPS control at these concentrations: 22 mM (P=0.0021), 11 mM (P=0.0017), 5.5 mM (P=0.0052) and 2.25 mM (P=0.0196). Besides, IL-6 secretion decreased compared with placebo at 22 mM (P=0.0177) and 11 mM (P=0.0031). CONCLUSION: The results indicate that the tested product exerts significant anti-inflammatory effects on human LPS-stimulated monocytes.

Successful treatment of an intrathoracic bronchogenic cyst in a Holstein-Friesian calf.

A 5-½-month-old female Holstein-Friesian calf was presented with a history of recurring ruminal tympany and poor development. The absence of lung sounds on the right hemithorax suggested a right-sided intrathoracic pathology. Radiography and computed tomography revealed a large thin-walled cavernous lesion with a gas-fluid interface which almost completely filled the right thoracic cavity. Fluid aspirated from the lesion was clear, yellowish and odorless. These findings led to the diagnosis of a bronchogenic cyst. Thoracotomy was performed under general anesthesia. The cyst strongly adhered to the adjacent lung tissue. After removal of the free wall, the adjacent lung tissue was sealed using surgical stapling instruments, and the non-removable part of the wall was curetted and rinsed. The intensive postoperative management included antibiotic therapy, oxygen supplementation and regional lidocaine infusion. Anti-inflammatory drugs were administered for further pain control. The calf recovered well and was released from the clinic on postoperative day 11. Intra- or extrathoracic bronchogenic cysts result from abnormal budding during the embryonic development of the tracheobronchial system. Successful treatment of this calf despite the size of the lesion and the invasive character of the surgical intervention indicates that resection of bronchogenic cysts in cattle may be an option for valuable animals.

Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis.

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

Ultrasonographic assessment of umbilical disorders.

Umbilical disorders are of great clinical relevance in calves during the early postnatal period. They may be classified as (1) noninfectious disorders such as hernias and urachal cysts, (2) infectious disorders involving extra- and intra-abdominal umbilical structures, or (3) combinations thereof. Supplementing clinical examination, umbilical ultrasonography allows the identification of the structures involved and differentiation of the various disorders with a high diagnostic sensitivity. A specific diagnosis of the umbilical disorder is important, because the treatment regimen, prognosis, and treatment costs completely depend upon the extent of the disease and the structures involved.

Ultrasonographic findings in 2 cows with duodenal obstruction.

Two cases where clinical findings suggestive of proximal ileus resulting in hypokalemic and hypochloremic alkalosis are presented. Ultrasonographs showed distension of the sigmoid flexure along the ventral border of the liver and between the serosal lamellae of the greater omentum in the flank, allowing exact preoperative obstruction localization.