Alexey Vanichkin

Ferrocene-induced lymphocyte activation and anti-tumor activity is mediated by redox-sensitive signaling

Ferrocene, a stable, synthetic, iron‐containing compound induces in vitro and in vivo activation of mouse lymphocytes and macrophages. Ferrocene also has a marked antitumor effect in mice, upon its administration intraperitoneally and in drinking water. Ferrocenes antitumor activity is attributed to its immune‐stimulatory property. This conclusion is supported by adoptive transfer experiments demonstrating that immune cells from ferrocene‐treated tumor‐bearing mice elicit an antitumor effect in mice not treated with ferrocene. We postulate that the immune stimulatory effect of ferrocene is mediated by redox‐sensitive signaling such as activation of p21ras. This postulation is supported by the following findings: Ferrocene generates H2O2 by autooxidation; N‐acetylcysteine, a free‐radical scavenger, reduces its antitumor effect; and it stimulates GTPase activity catalyzed by pure recombinant p21ras and activates ERK 1/2 in wild Jurkat T cells but fails to do so in the Jurkat T cells expressing p21ras in which cysteine 118 was replaced by serine. Lastly, ferrocene activates and translocates NF‐kB in human PBM, a pathway which is mediated by ras. It is most plausible that additional redox‐sensitive signaling proteins mediate the biological effects of ferrocene.

Vascularization of wide pore agarose–gelatin cryogel scaffolds implanted subcutaneously in diabetic and non-diabetic mice

Polymeric scaffolds have been reported to promote angiogenesis, facilitating oxygen delivery; however, little is known about the effect of diabetes on the neo-vascularization of implanted polymeric scaffolds at subcutaneous (SC) sites. In this study we compare the effect of diabetes on scaffold vascularization following SC implantation into diabetic and non-diabetic mice. Wide pore agarose cryogel scaffolds with grafted gelatin were prepared by a two-step freezing procedure and subsequent thawing. The scaffolds were implanted subcutaneously into streptozoticin-induced diabetic mice and control, non-diabetic mice. The vascularization process was estimated using histological sections, in which endothelial cells were identified by Von Willebrand factor (vWF) and CD31 antigen staining and the pericyte layer was confirmed by alpha-smooth muscle actin (alpha-SMA) visualization. Comparative analysis showed a similar thickness of fibrous capsules around the vascularized scaffolds in both diabetic and non-diabetic animals. Intensive staining for alpha-SMA indicated the formation of mature blood vessels in the surrounding fibrous capsule and tissue invading the scaffold area. No statistically significant differences in capillary density and area occupied by blood vessels were found between diabetic and non-diabetic mice. In conclusion, the present study shows no adverse effects of diabetes on new blood vessel formation in SC implanted agarose cryogel scaffolds with grafted gelatin.

Islet transplantation in a subarachnoid cavity surrounding olfactory bulb of diabetic rats.

Olfactory Bulb of Diabetic Rats C linical trials indicate that islet transplantation is a potential successful treatment for patients with type 1 and a subgroup of patients with type 2 diabetes. Usually, the liver is used as a site for islet transplantation. However, islet transplantations in this site are hampered by insufficient oxygenation, cell rejection, and procedure-related complications. The subarachnoid cavity, filled with cerebrospinal fluids, was considered as a potential site for cell transplantation because of immunoprivileged properties, excellent oxygen and nutrition supply, efficient metabolic waste removal, relatively large volume, and easy access. However, little has been published on the islet transplantation in this site. In the past, normoglycemia has been achieved in diabetic rats after syngeneic and allogeneic islet transplantation in the cerebrospinal fluids of cerebral ventricles (1, 2) or spinal cord (3). In this study, we investigated glucose homeostasis in diabetic rats after islet transplantation in a subarachnoid cavity surrounding the olfactory bulb. The olfactory bulb is known to be enriched with insulin receptor, displayed the highest transport rate for insulin in the brain (4), and plays an important role in the development of different neurodegenerative diseases, particularly in Alzheimer and Parkinson diseases (5). It has been shown that decreased brain insulin levels and/or signaling are associated with impaired learning, memory, and various neurodegenerative diseases (6). A recent clinical trial has indicated that intranasal delivery of insulin through the olfactory pathway may constitute a promising therapy for diabetesand age-related neurodegenerative disorders (7). With this rationale, we studied the different parameters of glucose homeostasis and islet morphology after islet transplantation in a subarachnoid cavity of diabetic rats (see Materials and Methods). This transplantation procedure enabled the assembly of grafted islets directly onto the glomeruli of the olfactory bulb. As shown in Figure 1A, all severely diabetic rats transplanted with 3000 islet equivalents achieved normoglycemia within the first 2 days, which was maintained for 2 months after transplantation. One month and two months after transplantation, the rats demonstrated normal glucose tolerance typical for healthy animals (Fig. 1B). These data are well correlated with the normalization of blood C-peptide level found in transplanted animals (mean [SD], 526 [90], 56 [47], and 372 [129] pmol/L in intact, diabetic, and transplanted rats, respectively). In contrast, the nontransplanted diabetic animals were permanently hyperglycemic, showing impaired glucose tolerance, decreased level of blood C-peptide, and body weight loss. They died within the first month after diabetes induction.

Involvement of prostaglandins in an animal model of Shigella-related seizures

We investigated whether prostaglandins (PGs), proinflammatory mediators implicated in excitatory activity, are involved in Shigella-related seizures. Pretreatment with S. dysenteriae sonicate (2LD(50)) enhanced mice response to pentylenetetrazole-induced seizures, without increase of brain concentrations of PGE(2), PGD(2) or PGF(2alpha). Preinjection of NS-398, an inhibitor of cyclooxygenase-2, before treatment with Shigella sonicate, had no effect on seizures. The anticonvulsive PGD(2) increased after injection of 8 LD(50) of Shigella sonicate, which did not enhance seizures (32 pg/mg vs 26 pg/ml, p=0.0063). The findings indicate that PGs are not involved in the enhanced seizure response after exposure to Shigella. However, induction of PGD(2) may play an inhibitory role.

Insulin delivery to the brain using intracranial implantation of alginate‐encapsulated pancreatic islets

There is increasing evidence supporting a link between cognitive dysfunctions and impaired brain insulin signalling. Insulin therapy has previously been tested as an approach to ameliorate brain insulin resistance and deficiency in patients with various brain disorders. However, current strategies for insulin delivery to the brain may induce severe hypoglycaemia when injected peripherally or show poor uptake when delivered intranasally. Recently, we have shown that intracranial transplantation of naked pancreatic islets increased insulin content in the brain and attenuated cognitive dysfunctions without altering peripheral glucose homeostasis in rats with schizophrenia‐like syndrome. In this study, we show that intracranial implantation of 50 pancreatic islets encapsulated in disc‐shaped alginate is sufficient to elevate insulin content in the rat brain. Three weeks after implantation, the islets displayed intact morphology, intensive hormone staining and glucose‐sensitive insulin release. The ultrapure alginate with high guluronic acid content used for islet encapsulation demonstrated good biocompatibility and stability after intracranial implantation. All implanted animals were normoglycaemic and normoinsulinaemic. In conclusion, the intracranial implantation of a small amount of alginate‐encapsulated islets is an efficient tool for metabolically regulated insulin delivery to the brain. Copyright

Intracranial pancreatic islet transplantation increases islet hormone expression in the rat brain and attenuates behavioral dysfunctions induced by MK-801 (dizocilpine).

The treatment of rodents with non-competitive antagonist of the N-Methyl-D-aspartate (NMDA) receptor, MK-801 (dizocilpine), induces symptoms of psychosis, deficits in spatial memory and impairment of synaptic plasticity. Recent studies have suggested that insulin administration might attenuate the cognitive dysfunctions through the modulatory effect on the expression of NMDA receptors and on the brain insulin signaling. Intrahepatic pancreatic islet transplantation is known as an efficient tool for correcting impaired insulin signaling. We examined the capacity of syngeneic islets grafted into the cranial subarachnoid cavity to attenuate behavioral dysfunctions in rats exposed to MK-801. Animals were examined in the open field (OF) and the Morris Water Maze (MWM) tests following acute or subchronic administration of MK-801. We found well-vascularized grafted islets expressing insulin, glucagon and somatostatin onto the olfactory bulb and prefrontal cortex. Significantly higher levels of insulin were detected in the hippocampus and prefrontal cortex of transplanted animals compared to the non-transplanted rats. All animals expressed normal peripheral glucose homeostasis for two months after transplantation. OF tests revealed that rats exposed to MK-801 treatment, showed hyper-responsiveness in motility parameters and augmented center field exploration compared to intact controls and these effects were attenuated by the grafted islets. Moreover, in the MWM, the rats treated with MK-801 showed impairment of spatial memory that were partially corrected by the grafted islets. In conclusion, intracranial islet transplantation leads to the expression of islet hormones in the brain and attenuates behavioral and cognitive dysfunctions in rats exposed to MK-801 administration without altering the peripheral glucose homeostasis.

4-Nitrobenzylidene malononitrile reduces apoptosis-mediated liver injury in mice.

BACKGROUND Apoptosis plays a role in experimental and clinically related liver damage. Inhibitors of tyrosine kinases were shown to modulate apoptosis induced by different agents in various cell types. AIMS Investigation of the effect of 4-nitrobenzylidene malononitrile (belonging to the tyrphostins family which are selective inhibitors of protein tyrosine kinases) on apoptosis-mediated acute liver injury. METHODS Two murine experimental models exhibiting apoptosis-mediated liver injury were used: (1) mice treated with tumor necrosis factor-alpha and D-galactosamine; and (2) mice treated with anti-Fas antibody. Liver injury was assessed by serum levels of transaminases and by microscopic analysis. Apoptosis was assessed by labeling of apoptotic cells in the liver by the TUNEL assay and by determination of caspase-3 activity. RESULTS Pretreatment of mice with 4-nitrobenzylidene malononitrile reduced tumor necrosis factor-alpha/D-galactosamine-induced hepatotoxicity. TUNEL positive cells in sections from livers treated with vehicle (control), 4-nitrobenzylidene malononitrile, tumor necrosis factor-/d-galactosamine and tumor necrosis factor-alpha/D-galactosamine and 4-nitrobenzylidene malononitrile, were >0.2, >0.2, 49+/-2.3 and 4+/-0.2 per mm(2), respectively. 4-Nitrobenzylidene malononitrile also reduced hepatotoxicity induced by anti-Fas antibody. Caspase-3 activation induced by either tumor necrosis factor-alpha/D-glactosamine or by anti-Fas treatment, was reduced by pretreatment with N-nitrobenzylidene malononitrile. CONCLUSIONS The findings may provide a base for development of a new therapeutic modality to reduce apoptosis-mediated liver damage.

Intracerebroventricular Streptozotocin Induces Obesity and Dementia in Lewis Rats

BACKGROUND Animal models of dementia associated with metabolic abnormalities play an important role in understanding the bidirectional relationships between these pathologies. Rodent strains develop cognitive dysfunctions without alteration of peripheral metabolism following intracerebroventricular administration of streptozotocin (icv-STZ). OBJECTIVE We aimed to estimate the effect of icv-STZ on cognitive functions and peripheral metabolism in Lewis rats, which are rarely used for the induction of cognitive abnormalities. METHODS Inbred adult Lewis rats were treated with single icv-STZ (3 mg/kg). Cognitive functions were assessed using Morris water maze (MWM) test and locomotion by the Open Field test. Metabolic alterations were studied using histological and biochemical analysis of brain and peripheral tissues. RESULTS The icv-STZ induced rapid weight decline during the first two weeks. Thereafter, the rats showed an accelerated weight gain. Three months after the icv-STZ treatment, the rats were severely obese and revealed fatty liver, pancreatic islet hypertrophy, significantly elevated levels of blood insulin, leptin, and adiponectin, but intact peripheral glucose homeostasis. The icv-STZ rats expressed amyloid-β deposits in blood vessels of leptomeningeal area, microgliosis, astrogliosis, and spongiosis in fimbria-fornix area of hippocampus. Locomotor activities of icv-STZ treated and sham-operated rats were similar. In the MWM test, the icv-STZ treated rats demonstrated severely impaired spatial learning during both acquisition and reversal phases. CONCLUSIONS Icv-STZ treated Lewis rats develop severe dementia associated with obesity and peripheral metabolic abnormalities. This animal model may be useful for exploring the pathophysiological relationship between obesity and dementia and provides a new tool for development of effective therapy.

INTRACRANIAL PANCREATIC ISLET TRANSPLANTATION ATTENUATES COGNITIVE DYSFUNCTIONS IN RAT MODELS OF ALZHEIMER’S DISEASE INDUCED BY ICV-STREPTOZOTOZIN (STZ) ADMINISTRATION, AND IN RAT EXPOSED TO DIZOCILPINE (MK-801)

or “connectors” on ascertainment efficiency. Methods:The Center for Outreach in Alzheimer’s, Aging, and Community Health (COAACH) is a well-established community resource, located at a Historically Black University, which provides education and ongoing support for AD patients and families. Anecdotal observation revealed that individuals who engaged in its community activities were willing to serve as “connectors” to other family members to facilitate their participation in community events and study participation. To formally assess their impact, we compared ascertainment outcomes in our families with “connectors” (N1⁄412) vs. those without “connectors” (N1⁄43). Primary outcomes consisted of numbers of recruitment calls, samples collected, and family members enrolled. Results: Preliminary data show that having a family member as a “connector” to facilitate study activities yielded much improved ascertainment outcomes. In families with a “connector”, fewer calls were made per family (mean1⁄4 2.0) than those without a “connector (mean 1⁄4 4.3). In all instances, the two calls to families with “connectors” were for referral followed by scheduling. In addition, we enrolled and sampled 72% of the eligible family members from families with a “connector” vs 27% in those families without a “connector.” Conclusions:Our preliminary data support the concept of engaging AA families in culturally relevant education to improve recruitment for studies in AD. Community engagement, in combination with identifying a family “connector”, who influenced other family members’ engagement, improved recruitment of multiplex families for a genomics study. This strategy may have significant impact on ascertainment efficiency and reduced recruitment costs. Improving recruitment efficiency is especially important as increasing numbers of studies enroll multiplex families from minority populations for genomic studies of AD.