Sławomir Wójcik

Crosstalk between autophagy and proteasome protein degradation systems: possible implications for cancer therapy.

The Ubiquitin-Proteasomes System (UPS) and autophagy, two main intracellular protein degradation pathways within the eukaryotic cells which were originally regarded as rather independent, seem to be very closely related. Proteasome inhibitors, including the multipathway inhibitor bortezomib, are drawing increased attention for their therapeutic potential in the treatment of chronic inflammation and cancer, especially tumours with a high degree of malignancy. The over-activation of autophagy induces cell death and may act as a powerful tumour-suppressing mechanism. However, autophagy, serving as an important mechanism to generate nutrients in time of cellular stresses, may directly contribute to the survival of cells treated with proteasome inhibitors, and in consequence, may decrease the effectiveness of therapy. Results of studies performed on several cancer cell lines demonstrated synergy between proteasome inhibitors and autophagy inhibitors. Those results became the base for ongoing clinical trials investigating autophagy inhibition in combination with anti-cancer therapies, including bortezomib. This review provides summary of the latest data on the functioning of the UPS and the mechanisms of autophagy. The new insights describing the main pathways of autophagy activation in response to UPS inhibition related to: (i) Unfolded Protein Response, (ii) PI3K/Akt/mTOR pathway, and (iii) formation of aggresomes, are discussed. It is concluded that concomitant inhibition of the two main cellular protein degradation systems may provide new therapeutic modalities for cancer treatment.

Canine inflammatory myopathy associated with Leishmania Infantum infection

Inflammatory myopathy associated with several infectious diseases occurs in dogs including those caused by Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Hepatozoon canis. However, muscle disease due to Leishmania infection has been poorly documented. The aim of this study was to examine the distribution and types of cellular infiltrates and expression of MHC class I and II in muscle biopsies obtained from 15 male beagle dogs from a breeder group with an established diagnosis of leishmaniasis. Myopathic features were characterized by necrosis, regeneration, fibrosis and infiltration of mononuclear inflammatory cells consisting of lymphocytes, plasma cells and histiocytes. The predominant leukocyte populations were CD3+, CD8+ and CD45RA+ with lesser numbers of CD4+ cells. Many muscle fibers had MHC class I and II positivity on the sarcolemma. There was a direct correlation between the severity of pathological changes, clinical signs, and the numbers of Leishmania amastigotes. Our studies provided evidence that: 1) Leishmania should be considered as a cause of IM in dogs; 2) Leishmania is not present within muscle fibers but in macrophages, and that 3) the muscle damage might be related to immunological alterations associated with Leishmania infection. Leishmania spp. should also be considered as a possible cause in the pathogenesis of human myositis.

Syrian hamster infected with Leishmania infantum: A new experimental model for inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are inflammatory disorders of unknown origin. On the basis of clinical, histopathological, and immunological features, they can be differentiated into three major and distinct subsets: dermatomyositis; polymyositis; and inclusion‐body myositis. Although a few animal models for IIM are currently available, they lack several characteristic aspects of IIMs. The aim of our study was to examine skeletal muscle involvement in an experimental animal model of visceral leishmaniasis, a disseminated infection caused by the protozoan parasite Leishmania infantum, and to compare features of associated inflammation with those of human IIM. Syrian hamsters infected intraperitoneally with amastigotes of L. infantum were killed at 3 or 4 months post‐infection, and the skeletal muscles were studied. Focal inflammation was predominantly observed in the endomysium and, to a lesser extent, in perivascular areas. Degenerating muscle fibers were also found, as well as myonecrosis. Immunofluorescence with confocal laser scanning microscopy was used to characterize the phenotype of inflammatory infiltrates and the distribution of MHC class I and II in muscle biopsies. The infiltrating inflammatory cells consisted mainly of T cells, and CD8+ T cells were found in non‐necrotic muscle fibers that expressed MHC class I on the sarcolemma. In addition to T cells, several macrophages were present. The model we are proposing closely resembles polymyositis and may be useful in studying certain aspects of this disease such as the role of T cells in muscle inflammation and myocytotoxicity, while also providing novel therapeutic targets. Muscle Nerve, 2009

Colocalization of neuropeptides with calcium-binding proteins in the claustral interneurons during postnatal development of the rat

The claustrum is a relatively large telencephalic structure, situated close to the border of the neo- and allocortical regions. Its neuronal population consists of glutamatergic, projecting neurons and GABA-ergic interneurons, characterized by occurrence of numerous additional biochemical markers. The postnatal development of these latter neurons has not been extensively studied. Revealing the characteristic patterns of colocalizations between selected markers may shed some light on their function and origin. We investigated the colocalization patterns between three neuropeptides: neuropeptide Y, somatostatin, vasoactive intestinal polypeptide and three calcium-binding proteins: calbindin D28k, calretinin, parvalbumin in the interneurons of the rat claustrum during a four-month postnatal period (P0-P120; P: postnatal day). Our studies revealed the following types of colocalizations: neuropeptide Y with calbindin D28k, calretinin or parvalbumin; somatostatin with calbindin D28k; vasoactive intestinal polypeptide with calretinin. Only vasoactive intestinal polypeptide- and calretinin-containing, double-labeled neurons were present at the day of birth, whereas the other double-labeled neurons appeared at later stages of development. The ratios of colocalizing neurons to single-labeled neurons in each type of colocalization were differentiated and reached the highest value (51%) for vasoactive intestinal polypeptide- and calretinin-double-labeled neurons. In conclusion, the claustral interneurons represent differentiated population in respect to the occurrence of neuropeptides and calcium-binding proteins. The expression of studied substances is changing during the postnatal period.

Postnatal development of the basolateral complex of rabbit amygdala: a stereological and histochemical study

The aim of the study was to estimate developmental changes in the rabbit basolateral complex (BLC) by stereological and histochemical methods. Material consisted of 45 brains of New Zealand rabbits (aged from 2 to 180 days, P2 to P180) of both sexes, divided into nine groups. The following parameters were estimated: volume of the cerebral hemisphere; volume of the whole BLC and of particular BLC nuclei; neuronal density and total number of neurons in these nuclei. Developmental changes in acetylcholinesterase (AChE) activity in the BLC were also examined. The volume of the cerebral hemisphere increased until P30, whereas volumes of nuclei increased for longer – until P90. The density of neurons in all nuclei studied reached the level characteristic for an adult animal at about P30. The total number of neurons in the dorsolateral division of the lateral nucleus (Ldl) stabilized the earliest – between P30 and P60, whereas in the ventromedial division of the lateral nucleus (Lvm), basomedial (BM) and basolateral (BL) nuclei the number stabilized later – between P60 and P90. AChE activity appears minimal in the BLC on P2, reaches a maximum on P30 and then decreases to the level characteristic of an adult animal on P60. AChE activity was greater in BL than in other nuclei in all age groups. Reaching adult AChE activity 1 month earlier than the total number of neurons in the BLC may indicate a role of the cholinergic system in BLC maturation.

Quantitative analysis of influence of sevoflurane on the reactivity of microglial cells in the course of the experimental model of intracerebral haemorrhage

Backgrounds: Microglial cells play an important role in the pathophysiology of intracerebral haemorrhage. We have examined the possible influence of sevoflurane on the reactivity of microglial cells during intracranial haemorrhage. Methods: Forty adult male rats were divided into two groups. All animals were anaesthetized with fentanyl, dehydrobenzperidol and midazolam. In the experimental group animals additionally received sevoflurane 2.2 vol% end‐tidal concentration. Intracranial haemorrhage was produced through infusion of blood into the striatum. The microglial cell population (numerical density of immunoreactive cells and their distribution) was assessed on days 1, 3, 7, 14 and 21 after producing a haematoma using antibodies OX42 and OX6. Results: In the control group significant differences in the density of OX42‐ir cells between 3rd and 7th (81.86 vs. 129.99) (95% CI: −77.99 to −18.25, P = 0.0035) and between 14th and 21st (105.36 vs. 63.81) (95% CI: 13.21 to 69.89, P = 0.006) survival days were observed. However, significant increase of percentage of amoeboid OX42‐ir cells between 3rd and 7th (0.98 vs. 48.71) (95% CI: −52.17 to −43.30, P = 0.0001) and between 7th and 14th (48.71 vs. 58.47) (95% CI: −13.96 to −5.55, P = 0.0002) and then their decrease – between 14th and 21st (58.47 vs. 31.74) (95% CI: 22.52 to 30.93, P = 0.0001) days of observation were noted. In the sevoflurane groups OX42‐ir cells were not found. On the 3rd day the density of OX6‐ir cells in the sevoflurane group was significantly lower than that in the control group (12.39 vs. 34.57) (95% CI: −49.78 to −2.96, P = 0.02). The percentage of an amoeboid form of OX6‐ir cells was significantly lower in the sevoflurane group than that in the control group (27.31 vs. 82.03) (95% CI: −72.52 to −36.92, P = 0.0001) (58.76 vs. 82.37) (95% CI: −38.81 to −8.41, P = 0.003) (42.87 vs. 81.55) (95% CI: −53.23 to −24.10, P = 0.0001) respectively for 3rd, 7th and 14th days of survival. Conclusion: Administration of sevoflurane during anaesthesia in animals with intracerebral haemorrhage evoked a decrease of activation of the microglial cells.

Age-Related Changes in Skeletal Muscle of Cattle

Sarcopenia, the age-related loss of muscle mass and strength, is a multifactorial condition that represents a major healthcare concern for the elderly population. Although its morphologic features have been extensively studied in humans, animal models, and domestic and wild animals, only a few reports about spontaneous sarcopenia exist in other long-lived animals. In this work, muscle samples from 60 healthy Podolica-breed old cows (aged 15–23 years) were examined and compared with muscle samples from 10 young cows (3–6 years old). Frozen sections were studied through standard histologic and histoenzymatic procedures, as well as by immunohistochemistry, immunofluorescence, and Western blot analysis. The most prominent age-related myopathic features seen in the studied material included angular fiber atrophy (90% of cases), mitochondrial alterations (ragged red fibers, 70%; COX-negative fibers, 60%), presence of vacuolated fibers (75%), lymphocytic (predominantly CD8+) inflammation (40%), and type II selective fiber atrophy (40%). Immunohistochemistry revealed increased expression of major histocompatibility complex I in 36 cases (60%) and sarcoplasmic accumulations of β-amyloid precursor protein–positive material in 18 cases (30%). In aged cows, muscle atrophy was associated with accumulation of myostatin. Western blot analysis indicated increased amount of both proteins—myostatin and β-amyloid precursor protein—in muscles of aged animals compared with controls. These findings confirm the presence of age-related morphologic changes in cows similar to human sarcopenia and underline the possible role of amyloid deposition and subsequent inflammation in muscle senescence.

Age related skeletal muscle atrophy and upregulation of autophagy in dogs

Sarcopenia, the age related loss of muscle mass and strength, is a multifactorial condition that occurs in a variety of species and represents a major healthcare concern for older adults in human medicine. In veterinary medicine, skeletal muscle atrophy is often observed in dogs as they reach old age, but the process is not well understood. Autophagy is a mechanism for degradation and recycling of cellular constituents and is potentially involved in sarcopenia. The aim of the present study was to evaluate the expression of three markers of autophagy, Beclin 1, LC3 and p62, in muscle wasting of geriatric dogs, to establish whether the levels of autophagy change with increasing age. Muscle biopsies from 25 geriatric dogs were examined and compared with those from five healthy young dogs. Samples from older dogs, assessed by routine histology, histoenzymatic staining and immunohistochemistry, showed evidence of muscle atrophy, sarcoplasmic vacuolisation and mitochondrial alterations. Furthermore, in 80% of the muscle samples from the older dogs, marked intracytoplasmic staining for Beclin 1 and LC3 was observed. Significantly greater expression of LC3 II and Beclin 1, but lower expression of p62, was found by Western blotting, comparing muscle samples from old vs. young dogs. The results of the study suggest that enhanced autophagy might be one of the factors underlying muscle atrophy in dogs as they age.

NPY-, SOM- and VIP-containing interneurons in postnatal development of the rat claustrum.

A growing body of evidence indicates the common origin of the claustrum, endopiriform nucleus, and the basolateral nuclear complex of amygdala from the lateral and ventral parts of the pallium, as the claustroamygdaloid complex. It seems very probable that at least some of the claustral interneurons derive from subcortical sources. The postnatal development of neuropeptide Y-, somatostatin- and vasoactive intestinal polypeptide-containing interneurons was studied during the 4 postnatal months (P0-P120; P, postnatal day). The study was conducted on 45 Wistar rats of both sexes. Our results indicate that neuropeptide-containing interneurons are not morphologically mature at the moment of birth. The characteristic features of neuronal bodies and the relatively long period of postnatal development may indicate their migration from the subcortical neurogenetic centers. Morphological changes in the neuropil are also reported. Although developmental patterns differ between various neuropeptide-containing neuronal subpopulations, two phases of development can be distinguished in each of them: the early phase (P0-P4) during which undifferentiated neurons and neuropil dominate, and the late phase (P7-P28) during which the characteristic features of an adult-like structure gradually appear. Later these observed developmental changes are terminated. The postnatal development of neuropeptide-containing interneurons is completed after 4 weeks of life. This period, which is important for the structural and functional development of the claustrum, must be taken into account in future studies on this structure.

Qualitative and quantitative study of the postnatal development of the rabbit claustrum.

The morphometric analysis of changes occurring in the rabbit claustrum in the early postnatal period was performed by means of unbiased stereological methods. Material consisted of 40 animals aged from P2 to P180 (P—postnatal day) divided into eight groups. The volume of the claustrum, total number and numerical density of its neurons change very rapidly at the beginning of the postnatal life and stabilize by about the fourth week.