Postpartum development of metabolic dysfunction-associated steatotic liver disease in a lean mouse model of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is associated with increased postpartum risk for metabolic dysfunction-associated steatotic liver disease (MASLD). GDM-related MASLD predisposes to advanced liver disease, necessitating a better understanding of its development in GDM. This preclinical study evaluated the MASLD development in a lean GDM mouse model with impaired insulin secretion capacity. Lean GDM was induced by short-term 60% high-fat diet and low-dose streptozotocin injections (60 mg/kg for 3 days) before mating in C57BL/6N mice. The control dams received only high-fat diet or low-fat diet. Glucose homeostasis was assessed during pregnancy and postpartum, whereas MASLD was assessed on postpartum day 30 (PP30). GDM dams exhibited a transient hyperglycemic phenotype during pregnancy, with hyperglycaemia reappearing after lactation. Lower insulin levels and impaired glucose-induced insulin response were observed in GDM mice during pregnancy and postpartum. At PP30, GDM dams displayed higher hepatic triglyceride content compared controls, along with increased MAS (MASLD) activity scores, indicating lipid accumulation, inflammation, and cell turnover indices. Additionally, at PP30, GDM dams showed elevated plasma liver injury markers. Given the absence of obesity in this double-hit GDM model, the results clearly indicate that impaired insulin secretion driven pregnancy hyperglycaemia has a distinct contribution to the development of postpartum MASLD.

The WNT receptor ROR2 drives the interaction of multiple myeloma cells with the microenvironment through AKT activation.

Multiple myeloma is the second most frequent hematological cancer after lymphoma and remains an incurable disease. The pervasive support provided by the bone marrow microenvironment to myeloma cells is crucial for their survival. Here, an unbiased assessment of receptor tyrosine kinases overexpressed in myeloma identified ROR2, a receptor for the WNT noncanonical pathway, as highly expressed in myeloma cells. Its ligand, WNT5A is the most abundant growth factor in the bone marrow of myeloma patients. ROR2 mediates myeloma cells interactions with the surrounding bone marrow and its depletion resulted in detachment of myeloma cells from their niche in an in vivo model, triggering apoptosis and thus markedly delaying disease progression. Using in vitro and ex vivo 3D-culture systems, ROR2 was shown to exert a pivotal role in the adhesion of cancer cells to the microenvironment. Genomic studies revealed that the pathways mostly deregulated by ROR2 overexpression were PI3K/AKT and mTOR. Treatment of cells with specific PI3K inhibitors already used in the clinic reduced myeloma cell adhesion to the bone marrow. Together, our findings support the view that ROR2 and its downstream targets represent a novel therapeutic strategy for the large subgroup of MM patients whose cancer cells show ROR2 overexpression.

Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

Intravesical Chemotherapy and Chemohyperthermia in Non-Muscle-Invasive Bladder Cancer; An Overview on Drug Administration Technologies and Pharmacokinetics.

BACKGROUND: Tumor recurrence is the most expected clinical event after the resection of non-muscle invasive bladder cancer, depending on histological findings of the initial lesion. In patients with low and intermediate risk of disease, the intravesical instillation of chemotherapy agents is recommended as a standard treatment to reduce recurrences. METHODS: A comprehensive review covering various aspects of different treatments with intravesical drugs is presented. RESULTS: Drugs may be instilled into the bladder starting with a single, 'early' postoperative administration or, after tumor resection with adjuvant intent or, before tumor resection under a neo-adjuvant regimen. Both latter protocols would consist of weekly treatments followed by monthly maintenance treatments. Different methods of administering drugs intravesically have been proposed to enhance the depth of drug penetration and its absorption into the bladder wall thus increasing the rate of drug-DNA reaction. These device-assisted therapies therefore have set a goal to potentiate the drug's effect and efficaciousness. The Radiofrequency-Induced Thermochemotherapeutic Effect (RITE) and the Electromotive-Drug Administration (EMDA) are the two most relevant modalities used to increase the activity of intravesical chemotherapy. Despite the widely adopted international guidelines' recommendations, and recent clinical trials of device-assisted chemotherapy instillations showing markedly enhanced recurrence-free survival compared even to the standard of care, clinicians and pharmacologists are not familiar with the in-depth physical aspects, pharmacokinetics and systemic absorption of chemotherapeutic drugs following their intravesical administration. CONCLUSION: Knowledge of drug diffusion mechanisms into the tissue and cellular cytoplasm following bladder instillation is a key to understand the safety profile and clinical activity of chemotherapy.

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 µM, 1 µM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Unexpected Cardiac Death During Anaesthesia of a Young Rabbit Associated with Fibro-fatty Replacement of the Right Ventricular Myocardium.

A 6-month-old female pet rabbit was presented for routine ovariectomy. The pre-anaesthetic evaluation was unremarkable and no anaesthetic complications occurred during the procedure. However, at the end of the surgery, the rabbit suddenly showed acute bradycardia and cardiac death. Necropsy examination revealed marked dilation of the right ventricle, associated with diffuse thinning of the right ventricular free wall. Gross and histopathological findings were suggestive of a congenital dilated cardiomyopathy characterized by fibro-fatty replacement of the right ventricular myocardium. Similar myocardial lesions have not been previously described in rabbits, although they have been documented in myocardial diseases of man, dogs, cats, cattle, horses and chimpanzees.

HSP32 and HSP90 Immunoexpression, in Relation to Kit Pattern, Grading, and Mitotic Count in Canine Cutaneous Mast Cell Tumors.

Literature data indicate heat shock protein (Hsp) 32 and 90 as potential molecular targets in canine neoplastic mast cells (MCs). However, their immunoexpression patterns in canine mast cell tumors (MCTs) have not been investigated. Thus, the aim of this study was to evaluate the immunohistochemical expression of Hsp32 and Hsp90 in 22 canine cutaneous MCTs, in relation to KIT immunolabeling pattern, histological grade, and mitotic count. All cases showed cytoplasmic labeling of Hsp90, variably associated with nuclear and/or membranous labeling. Relationships of Hsp90 or Hsp32 immunolabeling with KIT pattern, mitotic count, and tumor grade were not observed. However, the reduced Hsp32 immunoexpression observed in most grade III/high-grade MCTs suggests a tendency toward a loss of immunosignal in poorly differentiated MCs. The great heterogeneity in extent and distribution of Hsp90 immunoexpression among the different MCT cases may also partially explain the difficulties in predicting the in vivo biologic activity of Hsp90 inhibitors on canine MCTs.

CD3 and CD20 Coexpression in a Case of Canine Cutaneous Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides).

A 14-year-old female spayed Dachshund was presented with generalized scaling, erythema, pruritus, poor quality of hair coat, and progressive weight loss. Cutaneous epitheliotropic T-cell lymphoma (CETCL) was suspected. Skin biopsies were suggestive of CETCL. However, immunohistochemistry revealed the presence of numerous CD20+ and CD3+ cells. Clonality assay demonstrated a clonal T-cell receptor gamma rearrangement and a polyclonal IgH gene rearrangement. Double-label immunofluorescence confirmed coexpression of CD3 and CD20 by neoplastic cells. By double immunohistochemistry, neoplastic cells were CD3+ and PAX5-. The results are compatible with a CD3+, CD20+ CETCL. Coexpression of CD20 and CD3 has been recognized in peripheral T-cell lymphomas. Although documented in human CETCL, it has not been reported in canine CETCL. The pathogenetic basis of CD20 expression in mycosis fungoides is explored.

Immunohistochemical expression of heat shock proteins, p63 and androgen receptor in benign prostatic hyperplasia and prostatic carcinoma in the dog.

This study compared heat shock proteins Hsp60, Hsp72 and Hsp73, along with p63 and androgen receptor (AR) immunoexpression between 16 cases of benign prostatic hyperplasia (BPH) and 11 prostatic carcinomas (PCa) in dogs. The proportion of Hsp60-positive cells was higher in PCa compared with BPH (P = 0.033), whereas the frequency and intensity of Hsp73 immunostaining did not differ significantly between the two groups. Hsp72-immunostained nuclei formed a discontinuous layer along the basement membrane in BPH, whereas cells in this layer in PCa were negative or weakly positive. Hsp72 nuclear score showed significant positive associations with both p63 (P = 0.016) and AR (P = 0.009) scores. Double immunofluorescence revealed Hsp72-p63 and Hsp72-AR co-expressions in basal cell nuclei. Aberrant cytoplasmic p63 immunolabelling was observed in 3 of 11 PCa cases. These results suggest a role of the combined expression of Hsp72, p63 and AR in basal epithelial cells in canine BPH and PCa.

Survivin and related proteins in canine mammary tumors: immunohistochemical expression.

Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (ß-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy.

Immunohistochemical evaluation of heat shock protein expression in normal canine nerve and peripheral nerve sheath tumours.

Abnormal expression of heat shock proteins (HSPs) has been observed in many human neoplasms and such expression has prognostic, predictive and therapeutic implications. The aim of this study was to evaluate immunohistochemically the expression of HSP 27, HSP 32 and HSP 90 in normal canine peripheral nerves and in four benign and 15 malignant canine peripheral nerve sheath tumours (PNSTs). In normal nerve, all of the HSPs were detected in axons, epineurial fibroblasts and scattered Schwann cell bodies. Cytoplasmic expression of HSP 27 was more widespread and intense in benign PNSTs compared with malignant PNSTs (P <0.05). Widespread and intense nuclear expression of HSP 32 was also associated with benign tumours (P <0.01), while high HSP 90 immunoreactivity was detected in all tumours, suggesting that HSP 90 might represent a new therapeutic target.

Fixation-off sensitivity.

Fixation-off sensitivity (FOS) is a phenomenon induced by elimination of central vision/fixation, and may either manifest clinically with seizures or only represent an EEG abnormality. FOS is characterized by posterior or generalized epileptiform discharges that consistently occur after closing of the eyes and last as long as the eyes are closed. It is most commonly encountered in patients with idiopathic childhood occipital epilepsies, but may also be observed in cases of symptomatic or cryptogenic focal and generalized epilepsies, as well as in asymptomatic non-epileptic individuals. FOS should be differentiated from pure forms of scotosensitivity, in which EEG discharges or epileptic seizures are elicited by darkness, and from epileptiform discharges triggered by eye closure, which refer to eye closure sensitivity. Although FOS is probably associated with occipital hyperexcitability its intrinsic epileptogenic potential is presumed to be low.

Thermochemotherapy for non-muscle-invasive bladder cancer: is there a chance to avoid early cystectomy?.

OBJECTIVES: Non-muscle-invasive bladder cancer is characterized by a high recurrence rate after primary transurethral resection. In case of bacillus Calmette-Guérin-refractory neoplasms, cystectomy is the gold standard. In this study the effects of thermochemotherapy with mitomycin C were evaluated in high-risk bladder cancer nonresponders to previous therapy. PATIENTS AND METHODS: Between January 2006 and December 2009, 30 patients were enrolled with recurrent stage carcinoma in situ, Ta and T1, grade G1 to G3 non-muscle-invasive bladder cancer refractory to chemotherapy or immunotherapy and so becoming suitable for radical cystectomy. All patients underwent endovesical thermochemotherapy: 16 patients underwent a prophylactic scheme and 14 patients underwent an ablative scheme. RESULTS: All the patients completed the study. The mean follow-up for all the patients enrolled was 14 months. Thirteen of 30 patients (43.30%) were disease free and 17 patients (56.70%) had recurrence. In the prophylactic group, 7 of 16 patients (43.75%) were disease free and 9 patients (46.25%) had tumor recurrence; no progression was observed. In the ablative group, 3 patients (17, 64%) had progression to muscle-invasive disease. Side effects were generally mild. CONCLUSIONS: Thermochemotherapy could be considered an additional tool in patients refractory to intravesical therapies before considering early cystectomy.

IV Valproate in generalized convulsive status epilepticus: a systematic review.

Aim of this review was to evaluate efficacy and safety of intravenous valproate (IV VPA) in the treatment of generalized convulsive status epilepticus (GCSE) in patients of any age, synthesizing available evidences from randomized controlled trials (RCTs). RCTs on IV VPA administered in patients (no age restriction) for GCSE at any stage were searched in MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials. Studies were selected and data independently extracted. Following outcomes were considered: clinical seizure cessation after drug administration, seizure freedom at 24 h, and adverse effects. Outcomes were assessed using standard methods to calculate risk ratio (RR) with 95% confidence intervals. Five trials met inclusion criteria. Two different comparisons were available (IV VPA versus phenytoin (PHT), IV VPA versus IV Diazepam), but only the former included more than one study with enough information to permit a meta-analysis. Compared with PHT, VPA had statistically lower risk of adverse effects (RR 0.31, 95% CI 0.12-0.85), with no differences in GCSE cessation after drug administration (RR 1.31, 95% CI 0.93-1.84) and in seizure freedom at 24 h (RR 0.96, 95% CI 0.88-1.06). This review suggests that IV VPA has a better tolerability than PHT in treatment of GCSE, without any statistically significant differences in terms of efficacy. More rigorous RCTs of VPA versus an appropriate comparator, in a well-defined population with a systematic definition of SE, are however required to conclude about efficacy and tolerability of VPA in clinical practice.

ß-catenin in canine skin: immunohistochemical pattern of expression in normal skin and cutaneous epithelial tumours.

In normal adult skin, ß-catenin is a structural component of the intercellular junction and the Wnt/ß-catenin pathway plays a key role in the regulation of cutaneous homeostasis, particularly in the maintenance of hair follicle stem cells. No data are available on the expression pattern of ß-catenin in normal canine skin and in canine cutaneous epidermal and follicular tumours. The present study used immunohistochemistry to determine ß-catenin expression in four samples of normal canine skin and 62 cutaneous epithelial tumours (14 epidermal, 30 follicular and 18 glandular). ß-catenin expression was localized to the nucleus of matrical and dermal papilla cells in anagen hair follicles and was also found in scattered cells of the outer root sheath, suggesting that these follicular epithelial cells may have a high proliferative potential. Nuclear labelling, considered a hallmark of activation of the Wnt/ß-catenin signalling pathway, was observed in canine follicular tumours with matrical differentiation (100% of cases of trichoepithelioma and pilomatricoma), suggesting that a possible mutation of the canine CTNBB1 gene may underlie these tumours. In contrast, malignant tumours (squamous cell carcinoma, basal cell carcinoma, sebaceous and apocrine gland carcinoma and epithelioma) were characterized by reduction/loss of ß-catenin membrane labelling compared with normal cutaneous epithelial cells and benign tumours, suggesting that reduction/loss of ß-catenin expression is important in the acquisition of the malignant phenotype and may have a role in the infiltration and metastasis of these tumours.

Methodology of superficial bladder cancer trials: objective evaluation of treatment - the need for standardization.

The formulation of proper evaluation criteria after superficial bladder cancer therapy poses several methodological problems that are often peculiar to the disease. The Achilles' heel of many trials is possibly found in the criteria used in the evaluation of the trial's outcome. As a consequence, total agreement regarding the criteria for response and the evaluation of response is needed. The adoption of standard response criteria should be given high priority. Uniform criteria of response should be chosen because they meet standards of reliability and statistical validity. Thus, the criteria must be reproducible and correlate with some measures of patient benefit such as quantity and quality of survival. A proposal for standardization in superficial bladder cancer clinical trials is presented based upon the current knowledge of methodology used for conducting clinical trials and upon the experience coming from clinical research groups.

f-MRI in Epilepsy with Spike and Wave Activity Evoked by Eye Closure: Different Bold Activation in a Patient with Idiopathic Partial Epilepsy with Occipital Spikes and a Control Group.

We performed functional magnetic resonance imaging (fMRI) in a 30-year-old man with idiopathic partial epilepsy with occipital spikes whose scalp EEG activity was characterized by persistent epileptiform discharges on eye closure, ceasing upon eye opening. We compared BOLD activation in the patient and in a control group of three normal volunteers. f-MRI showed that occipital cortex and frontal areas were activated in relation to eye movement in normal subjects during eye opening but not during eye closing. While persistent interictal spike and wave activity was present over the posterior and anterior scalp in the patient upon eye closing, f-MRI showed bilateral activation of the parietal and temporal regions. This fMRI study documents the activation of posterior and temporal areas related to continuous intercritical spikes evoked by eye closure, which are diffuse over the scalp. This activation was absent in the control group during eye closure.

Continuous EEG-fMRI in Pre-Surgical Evaluation of a Patient with Symptomatic Seizures: Bold Activation Linked to Interictal Epileptic Discharges Caused by Cavernoma.

We used continuous electroencephalography-functional magnetic resonance imaging (EEG-fMRI) to identify the linkage between the "epileptogenic" and the "irritative" area in a patient with symptomatic epilepsy (cavernoma, previously diagnosed and surgically treated), i.e. a patient with a well known "epileptogenic area", and to increase the possibility of a non invasive pre-surgical evaluation of drug-resistant epilepsies. A compatible MRI system was used (EEG with 29 scalp electrodes and two electrodes for ECG and EMG) and signals were recorded with a 1.5 Tesla MRI scanner. After the recording session and MRI artifact removal, EEG data were analyzed offline and used as paradigms in fMRI study. Activation (EEG sequences with interictal slow-spiked-wave activity) and rest (sequences of normal EEG) conditions were compared to identify the potential resulting focal increase in BOLD signal and to consider if this is spatially linked to the interictal focus used as a paradigm and to the lesion. We noted an increase in the BOLD signal in the left neocortical temporal region, laterally and posteriorly to the poro-encephalic cavity (residual of cavernoma previously removed), that is around the "epileptogenic area". In our study "epileptogenic" and "irritative" areas were connected with each other. Combined EEG-fMRI may become routine in clinical practice for a better identification of an irritative and lesional focus in patients with symptomatic drug-resistant epilepsy.

Effects of sleep deprivation on cortical excitability in patients affected by juvenile myoclonic epilepsy: a combined transcranial magnetic stimulation and EEG study.

OBJECTIVE: To investigate the effect of sleep deprivation on corticospinal excitability in patients affected by juvenile myoclonic epilepsy (JME) using different transcranial magnetic stimulation (TMS) parameters. METHODS: Ten patients with JME and 10 normal subjects underwent partial sleep deprivation. Motor threshold (MT), motor evoked potential amplitude (MEP), and silent period (SP) were recorded from the thenar eminence (TE) muscles. Short latency intracortical inhibition (SICI) and short latency intracortical facilitation (SICF) were studied using paired magnetic stimulation. TMS was performed before and after sleep deprivation; EEG and TMS were performed simultaneously. RESULTS: In patients with JME, sleep deprivation induced a significant decrease in SICI and an increase in SICF, which was associated with increased paroxysmal activity. A significant decrease in the MT was observed. No significant changes in any TMS parameters were noted in normal subjects after sleep deprivation. The F wave was unchanged by sleep deprivation in both control subjects and in patients with JME. CONCLUSIONS: In patients with JME, sleep deprivation produces increases in corticospinal excitability in motor areas as measured by different TMS parameters.

Cutis verticis gyrata, mental retardation and Lennox-Gastaut syndrome: a case report.

Cutis verticis gyrata (CVG) is an abnormality of the scalp characterized by the formation of furrows and folds which cannot be flattened by traction or pressure. Primary and secondary forms of CVG have been described. We report on a patient affected by cutis verticis gyrata, mental regression and Lennox-Gastaut syndrome (LGS). Serum hormonal levels, karyotype and X fragile studies were normal. Magnetic resonance imaging of the brain showed only atrophic changes. The etiology of primary CVG remains unknown as does its relation with LGS.