Intraperitoneally administered IgG from patients with amyotrophic lateral sclerosis or from an immune-mediated goat model increase the levels of TNF-α, IL-6, and IL-10 in the spinal cord and serum of mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the selective loss of the upper and lower motor neurons (MNs). Neuroinflammation has been implicated in the pathogenesis of the sporadic form of the disease. We earlier developed immune-mediated animal models of ALS and demonstrated humoral and cellular immune reactions in the nervous system and in the sera of patients and animals. The accumulation of immunoglobulin G (IgG), an elevated intracellular level of calcium, ultrastructural alterations in the MNs, and activation of the microglia were noted in the spinal cord of ALS patients. Similar alterations developed in mice inoculated intraperitoneally with IgG from ALS patients or from an immune-mediated goat model. METHODS: We have now examined whether the intraperitoneal injection of mice with IgG from sporadic ALS patients or from immunized goats with the homogenate of the anterior horn of the bovine spinal cord is associated with changes in the pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines in the spinal cord and serum of the mice. The levels of cytokines were measured by ELISA. RESULTS: Intraperitoneally administered IgG from the ALS patients induced subclinical signs of MN disease, while the injection of IgG from immunized goats resulted in a severe respiratory dysfunction and limb paralysis 24 h after the injections. Significantly increased levels of TNF-α and IL-10 were detected in the spinal cord of the mice injected with the human ALS IgG. The level of IL-6 increased primarily in the serum. The IgG from the immunized goats induced highly significant increases in the levels of all three cytokines in the serum and the spinal cord of mice. CONCLUSIONS: Our earlier experiments had proved that when ALS IgG or IgG from immune-mediated animal models was inoculated into mice, it was taken up in the MNs and had the ability to initiate damage in them. The pathological process was paralleled by microglia recruitment and activation in the spinal cord. The present experiment revealed that these forms of IgG cause significant increases in certain cytokine levels locally in the spinal cord and in the serum of the inoculated mice. These results suggest that IgG directed to the MNs may be an initial element in the damage to the MNs both in human ALS and in its immune-mediated animal models.

GENETIC POLYMORPHISMS OF HUMAN ß-DEFENSINS IN PATIENTS WITH MULTIPLE SCLEROSIS.

AIMS: Recent studies have started to elucidate the contribution of microbiome to the pathogenesis of multiple sclerosis (MS). It is also supposed, that neuropathological alterations might be associated with abnormal expression and regulatory function of antimicrobial peptides (AMPs), including defensins. It is in our interest to investigate the relevance of the single nucleotide polymorphisms (SNPs) of the DEFB1 gene and the copy number polymorphism of the DEFB4 genes in MS. METHODS: DEFBI polymorphisms: c.-20G > A (rsl 1362), DEFB1 c.-44C > G (rsI 800972), DEFB1 c.-52G>A (rsl 799946), and the DEFB4 gene copy number were investigated in 250 MS patients The control patients comprised 232 age- and gender-matched healthy blood donors. The occurrence of the human ß-defensin 2 peptide (hBD2) in the plasma of controls and patients-was determined by ELISA. RESULTS: The DEFB1 c.-44C>G polymorphism the GG protective genotype was much less frequent among patients than among the controls. A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with MS as compared with the controls (43% vs. 28%, respectively). The median levels of the circulating hBD2 in the patients were 150.6 +/- 12.71 pg/ml vs. 262.1 +/- 23.82 pg/mI in the control group (p<0.0001). Our results suggest that ß-defensins play role in the development of MS.

The kynurenine system and immunoregulation.

There is developing interest in the role of the kynurenines in the immune function. A considerable amount of evidence has accumulated as concerns interactions between the kynurenine pathway, cytokines and the nervous system. Indoleamine 2,3-dioxygenase (IDO) occupies a key position connecting the immune system and the kynurenine pathway. There are evidences of the immunosuppressive effect of IDO. Following the interferon (IFN)-mediated activation of antigen presenting cells, the induction of IDO and the kynurenine system exerts a counter-regulating effect, maintaining the homeostasis. Inhibition of T cell functions, activation of the regulatory T cells, and the inhibition of Natural Killer cells are among the important factors in the immunosuppressive effects of IDO and kynurenines. There is a close connection between cytokines (IFN-α, IFN-γ, TNF-α, TGF-ß, IL-4 and IL-23) and the kynurenine system, and an imbalance in the TH1/TH2 cytokine profile may possibly lead to neurologic or psychiatric disorders. As the tryptophan metabolic pathway is activated by pro-inflammatory stimuli, the anti-inflammatory effect of kynurenic acid provides a further feedback mechanism in modulating the immune responses.

Immune Influencers in Action: Metabolites and Enzymes of the Tryptophan-Kynurenine Metabolic Pathway.

The tryptophan (TRP)-kynurenine (KYN) metabolic pathway is a main player of TRP metabolism through which more than 95% of TRP is catabolized. The pathway is activated by acute and chronic immune responses leading to a wide range of illnesses including cancer, immune diseases, neurodegenerative diseases and psychiatric disorders. The presence of positive feedback loops facilitates amplifying the immune responses vice versa. The TRP-KYN pathway synthesizes multifarious metabolites including oxidants, antioxidants, neurotoxins, neuroprotectants and immunomodulators. The immunomodulators are known to facilitate the immune system towards a tolerogenic state, resulting in chronic low-grade inflammation (LGI) that is commonly present in obesity, poor nutrition, exposer to chemicals or allergens, prodromal stage of various illnesses and chronic diseases. KYN, kynurenic acid, xanthurenic acid and cinnabarinic acid are aryl hydrocarbon receptor ligands that serve as immunomodulators. Furthermore, TRP-KYN pathway enzymes are known to be activated by the stress hormone cortisol and inflammatory cytokines, and genotypic variants were observed to contribute to inflammation and thus various diseases. The tryptophan 2,3-dioxygenase, the indoleamine 2,3-dioxygenases and the kynurenine-3-monooxygenase are main enzymes in the pathway. This review article discusses the TRP-KYN pathway with special emphasis on its interaction with the immune system and the tolerogenic shift towards chronic LGI and overviews the major symptoms, pro- and anti-inflammatory cytokines and toxic and protective KYNs to explore the linkage between chronic LGI, KYNs, and major psychiatric disorders, including depressive disorder, bipolar disorder, substance use disorder, post-traumatic stress disorder, schizophrenia and autism spectrum disorder.

Kynurenic Acid Analog Attenuates the Production of Tumor Necrosis Factor-α, Calgranulins (S100A 8/9 and S100A 12), and the Secretion of HNP1-3 and Stimulates the Production of Tumor Necrosis Factor-Stimulated Gene-6 in Whole Blood Cultures of Patients With Rheumatoid Arthritis.

Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with complex pathogenesis involving a variety of immunological events. Recently, it has been suggested that kynurenic acid (KYNA) might be a potential regulator of inflammatory processes in arthritis. KYNA has a definitive anti-inflammatory and immunosuppressive function. The aim of the present study is to investigate the complex effects of a newly synthesized KYNA analog-SZR72 on the in vitro production of tumor necrosis factor-α (TNF-α), tumor necrosis factor-stimulated gene-6 (TSG-6), calprotectin (SA1008/9), SA100 12 (EN-RAGE), and HNP1-3 (defensin-α) in the peripheral blood of patients with RA and the various effects of the disease. Methods: Patients with RA (n = 93) were selected based on the DAS28 score, medication, and their rheumatoid factor (RF) status, respectively. Peripheral blood samples from 93 patients with RA and 50 controls were obtained, and activated by heat-inactivated S. aureus. Parallel samples were pretreated before the activation with the KYNA analog N-(2-N, N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride. Following the incubation period (18 h), the supernatants were tested for TNF-α, TSG-6, calprotectin, S100A12, and HNP1-3 content by ELISA. Results: SZR72 inhibited the production of the following inflammatory mediators: TNF-α, calprotectin, S100A12, and HNP1-3 in whole blood cultures. This effect was observed in each group of patients in various phases of the disease. The basic (control) levels of these mediators were higher in the blood of patients than in healthy donors. In contrast, lower TSG-6 levels were detected in patients with RA compared to healthy controls. In addition, the KYNA analog exerted a stimulatory effect on the TSG-6 production ex vivo in human whole blood cultures of patients with RA in various phases of the disease. Conclusion: These data further support the immunomodulatory role of KYNA in RA resulting in anti-inflammatory effects and draw the attention to the importance of the synthesis of the KYNA analog, which might have a future therapeutic potential.

Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.

BACKGROUND: The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). METHODS: SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. RESULTS: Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma. CONCLUSION: These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.

Characteristic imprint of single nucleotide polymorphisms in multiple sclerosis.

Although the main pathomechanism of multiple sclerosis (MS) is not known, an autoimmune response against the myelin basic proteins (MBPs) is presumed to be involved in its evolution and propagation. In this study, we examined whether the nucleotide sequences of the 3' untranslated regions (UTRs) of the DNAs encoding the MBP are characteristic of MS. These genetic regions are presumed to be responsible for the transport and localization of the mRNAs encoding the MBP in the glia cells, thereby influencing the building up of the myelin sheaths of the glia cells. The DNA region involving nucleotides 710-1540 of the UTRs of the MBPs was sequenced and analyzed in 52 relapsing-remitting MS patients, in 52 neuroimaging alteration-free controls, and in 45 healthy volunteers. Although the examined UTRs exhibited a wide range of sequence variations in both the MS and the control subjects, we found a typical distribution of single nucleotide polymorphisms (SNPs) along the examined DNA sequence in the MS patients, which was different from that in the controls. We could distinguish two genetic regions: region A--nucleotide positions 851-896 and B--nucleotide positions 897-1540, in the UTRs. Subjects with SNPs in region A but without SNPs in region B occurred significantly more frequently in the MS group than in the control group (30.8% versus 3.85%, p < 0.0002848, OR 11.11, 95% CI--2.4-51.4, p < 0.0004). The distribution pattern of the SNPs in the UTRs seems to be highly characteristic of relapsing-remitting MS. These findings call attention to the possible roles of the UTRs of the MBPs in the development of MS.

Potential role of human beta-defensin 1 in Helicobacter pylori-induced gastritis.

OBJECTIVE: Helicobacter pylori-induced gastric inflammation is dependent on the persistence of the microorganism in the gastric epithelium. Modulation of the host epithelial antimicrobial responses may be a critical determinant in H. pylori-induced gastritis. Human beta-defensins (hBDs) are important components of the host defence at mucosal surfaces. The aim of the present study was to investigate the relevance of three single nucleotide polymorphisms (SNPs) of the human beta defensin-1 (hBD-1) gene in H. pylori-induced gastritis and to assess the mRNA expression of hBD-1 in H. pylori-infected AGS cells. MATERIAL AND METHODS: Three SNPs of the beta defensin DEFB1 gene, DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946), were genotyped either by Custom TaqMan SNP genotyping assays or by restriction fragment length polymorphism (RFLP) in 150 patients with chronic active gastritis; 100 serologically H. pylori-positive subjects without gastric or duodenal symptoms served as controls. hBD-1 mRNA expression in AGS cells was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Significant differences in frequencies of the GA and AA genotypes of G-52A SNPs were observed between patients with chronic active gastritis and healthy controls. The maximum level of hBD-1 mRNA expression in AGS cells was observed at 24 h after infection with H. pylori, this not being dependent on the presence of the cag pathogenicity island (PAI). CONCLUSIONS: The results of these genetic and in vitro experiments suggest that not only the inducible, but also the constitutive form of hBD may be important in the pathogenesis of H. pylori-induced gastritis.

Evaluation of the genetic variants of kinesin motor protein in ischemic stroke.

BACKGROUND: The kinesin light-chain 1 genetic variants G56836C, A185C, and C406T were earlier found to amplify the development of leukoaraiosis in hypertensive smokers. These 3 variants were presumed to affect the function of the mitochondria, thereby giving rise to sensitivity to a chronic ischemic state. We have now extended our investigations to examine how the above genetic variants affect the occurrence of ischemic stroke. METHODS: Genetic and clinical data on 650 ischemic stroke and 340 neuroimaging alteration-free subjects were analyzed. Univariate and logistic regression approaches were used. RESULTS: None of the above genetic variants proved to be risk factors of ischemic stroke, either alone or in combination with other clinical factors. CONCLUSION: The examined 3 genetic variants seem to influence the responses of the glial cells to a slight chronic hypoxia state, rather than the mechanisms resulting in cerebral infarcts themselves.

The Opposite Effects of Kynurenic Acid and Different Kynurenic Acid Analogs on Tumor Necrosis Factor-α (TNF-α) Production and Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) Expression.

Purpose: The investigation of anti-inflammatory and immunosuppressive functions of Kynurenic acid (KYNA) is now in focus. There is also substantial evidence that TSG-6 has an anti-inflammatory activity. Therefore, in the present study, we compared the effects of newly synthetized KYNA analogs on the TNF-α production in U-937 monocytic cells in correlation with the effects on the TSG-6 expression. Methods: TNF-α production was measured by ELISA, the TSG-6 expression was determined by RTqPCR method. As cytokine inducers Staphylococcus aureus and Chlamydia pneumoniae were used. Results: KYNA and KYNA analogs attenuated TNF-α production and increased TSG-6 mRNA expression in U-937 cells stimulated by heat inactivated Staphylococcus aureus. In contrast, KYNA and some of the KYNA analogs increased the TNF-α production of C. pneumoniae infected U-937 cells; however, the newly synthetized analogs (SZR104, SZR 105, and SZR 109) exerted significant inhibitory effects on the TNF-α synthesis. The inhibitory and stimulatory effects correlated inversely with the TSG-6 expression. Conclusions: TSG-6 expression following activation with bacterial components could participate in the suppression of inflammatory cytokines, such as TNF-α, We suppose that the elevation of the TSG-6 expression by KYNA and especially by new KYNA analogs might be one of the mechanisms that are responsible for their suppressive effect on TNF-α production as a feedback mechanism. KYNA and KYNA analogs have an important role in influencing TSG-6 expression, and there is a possible benefit of targeting TSG-6 expression by kynurenines in inflammatory conditions following infections.

A cytoskeleton motor protein genetic variant may exert a protective effect on the occurrence of multiple sclerosis: the janus face of the kinesin light-chain 1 56836CC genetic variant.

Although the main pathomechanism of multiple sclerosis (MS) is not known, an autoimmune response is presumed to involve its evolution and propagation. In this study, we examined how the kinesin light-chain 1 (KLC1) G56836C (rs8702) single nucleotide polymorphism (SNP) in intron 13 affects the occurrence of MS. This genetic variant was found to be associated with cognitive disturbances and neurodegeneration, and it was presumed to affect the kinesin function. Kinesin serves as a main cytoskeleton motor protein by carrying mitochondria and the molecular apparatus of myelin basic protein synthesis. The present association analysis of this genetic variant was performed in 102 relapsing-remitting MS patients and in 207 neuroimaging alteration-free controls. The KLC1 56836CC variant proved to exert a significant protective effect on the occurrence of MS (2.0% vs. 9.7%, P < 0.02; crude OR: 0.19, 95% CI: 0.04-0.82, P < 0.05; adjusted OR: 0.21, 95% CI: 0.018-0.88, P < 0.05). Our results draw attention to possible roles of the cytoskeleton in MS.

The risk of early and late lung sequelae after conformal radiotherapy in breast cancer patients.

PURPOSE: To study the risks of early and late radiogenic lung damage in breast cancer patients after conformal radiotherapy. METHODS AND MATERIALS: Radiogenic lung sequelae were assessed prospectively in 119 patients by means of clinical signs, radiologic abnormalities, and the mean density change (MDC) of the irradiated lung on CT. RESULTS: Significant positive associations were detected between the development of lung abnormalities 3 months or 1 year after the radiotherapy and the age of the patient, the ipsilateral mean lung dose (MLD), the radiation dose to 25% of the ipsilateral lung (D(25%)) and the volume of the ipsilateral lung receiving 20 Gy (V(20 Gy)). The irradiation of the axillary and supraclavicular lymph nodes favored the development of pneumonitis but not that of fibrosis. No relation was found between the preradiotherapy plasma TGF-beta level and the presence of radiogenic lung damage. At both time points, MDC was strongly related to age. Significant positive associations were demonstrated between the risks of pneumonitis or fibrosis and the age of the patient, MLD, D(25%), and V(20 Gy). A synergistic effect of MLD, D(25%), and V(20 Gy) with age in patients older than 59 years is suggested. CONCLUSION: Our analyses indicate that the risks of early and late radiogenic lung sequelae are strongly related to the age of the patient, the volume of the irradiated lung, and the dose to it.

A genetic variant in cytoskeleton motors amplifies susceptibility to leukoaraiosis in hypertensive smokers: gene-environmental interactions behind vascular white matter demyelinization.

One of the most frequent causes of an age-associated cognitive decline is the vascular white matter demyelinization of the brain referred to as leukoaraiosis (LA). The wide range of severity of the cognitive decline caused by LA can have numerous deleterious effects on the quality of life, leading overall to far-reaching public health problems. Besides clinical risk factors such as hypertension and advanced age, genetic susceptibility factors are presumed to be of great importance in the development of LA. The protein kinesin, which is the main motor protein in the trafficking system of the mitochondria, can undergo functional damage under the circumstances of chronic hypoxia. This may give rise to a slowly developing metabolic crisis in the glia cells, a phenomenon hypothesized to account for the evolution of LA. Setting out from this assumption, we examined how the kinesin light-chain 1 (KNS2) G56836C single nucleotide polymorphism in intron 13 affects the susceptibility to LA. This genetic variant was found to be associated with cognitive disturbances and neurodegeneration, and it was presumed to affect the function of kinesin. The association analysis of the above genetic variant was performed in 229 patients with LA and 264 neuroimaging alteration-free controls. The KNS2 56836CC variant increased the risk of LA 7.76-fold in hypertensive smokers as compared with those not carrying this variant. This finding may be useful in everyday clinical practice by indicating the need for stricter preventive measures in CC carriers.

Evaluation of the roles of the A185C and C406T kinesin light-chain 1 variants in the development of leukoaraiosis.

Vascular white matter demyelinization of the brain is referred to as leukoaraiosis (LA). This frequent age-associated entity leads to a cognitive decline or dementia. The background of LA has been hypothesized to be a chronic hypoxia-induced functional cytoskeleton malfunction. Setting out from this assumption, we earlier found that the kinesin light-chain 1 (KNS2) cytoskeleton motor protein 56836CC single nucleotide polymorphism conferred a risk of LA in hypertensive smokers. The aim of the present study was to extend our observations as to how the KNS2 A185C and C406T single nucleotide polymorphisms in the 5'-untranslated sequence region affect the susceptibility to LA. These two latter variants were presumed to influence the transcription of the KNS2 mRNA by locating in a function-enhancer region. An association analysis of these genetic variants was conducted on 242 patients with LA and 251 neuroimaging alteration-free controls. The KNS2 AA185-406TT haplotype increased the risk of LA 3.56-fold in hypertensive smokers as compared with those not carrying the KNS2 AA185-406TT genotype, which was similar to our previous findings for the KNS2 56836CC intron variant. Moreover, the three homozygous KNS2 variants (56936CC-AA185-406TT) coincided to an extent of 82.2%. Overall, although the 56836CC intron variant appears to be the most important of the three kinesin variants as regards the development of LA, the contribution of the AA185-406TT haplotype to the unfavorable phenotype of LA cannot be ruled out. The present finding supports the involvement of the cytoskeleton in the development of vascular white matter damage, thereby opening up novel fields in the research into LA.

Relevance of transforming growth factor-beta1, interleukin-8, and tumor necrosis factor-alpha polymorphisms in patients with chronic pancreatitis.

UNLABELLED: Cytokine regulation may be an important factor in the susceptibility for the development of chronic pancreatitis; transforming growth factor-beta1 (TGF-beta1) plays a central role in the pathogenesis of pancreatic fibrogenesis. The aim of our study was to analyse the relevance of TGF-beta1, interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) polymorphisms in patients with chronic pancreatitis. PATIENTS: of the 83 patients enrolled in the study, 43 were treated medically and 40 patients underwent surgical intervention. Healthy blood donors (n=75) served as controls. METHODS: the polymorphisms of TGF-beta1 +869 T--> C and IL-8 -251 T-->A were determined by the ARMS method, while that of TNF-alpha -308 was investigated using NcoI RFLP. RESULTS: there was a higher frequency (50%) of the TT genotype of TGF-beta1 +869, with a concomitantly higher TGF-beta1 level in the plasma (5.2 +/- 1.7 ng/mL) of patients with chronic pancreatitis than in healthy blood donors (28% and 2.8 +/- 0.9 ng/mL respectively). The number of TT homozygotes differed significantly between the patients who underwent surgical intervention and the controls, and even between the surgical and the non-surgical patients. The frequency of the T/A genotype with higher IL-8 production, was significantly higher in both groups of patients than in the controls (58% and 58% versus 40%). No correlation was found between the TNF-alpha -308 polymorphism and chronic pancreatitis. CONCLUSIONS: correlations of the TGF-beta1 and IL-8 single nucleotide polymorphisms (SNPs) with chronic pancreatitis underline the importance of these cytokines in the pathomechanism of the disease. Moreover, it seems that the TT genotype of +869 TGF-beta1 might be a risk factor for the development of a severe form of chronic pancreatitis, and could serve as a prognostic sign for any future surgical intervention or even repeat surgery. Further studies on a larger group of patients, in addition to a follow-up study, are necessary to confirm this preliminary observation.

Effects of nontoxic heat shock protein 90 inhibitor peptide derivatives on reversal of MDR of tumor cells.

Novel heat shock protein 90 inhibitor peptide derivatives [D- Trp-Phe-D- Trp-Leu-AMB (1), p-HOPA-D- TrpPhe-D-Trp-Leu-psi(CH2NH)-Leu-NH2 (2), D-Trp-Phe-D-Trp-OH (3), Suc-D-Trp-Phe-D-Trp-Leu-AMB (4), D-Tyr-Phe-D-Trp-Leu-AMB (5), D-Arg-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (6), Leu-psi(CH2NH)-Leu-NH2x2HCl (7), Phe-Trp-Phe-Trp-Leu-Leu-NH2 (8), Tyr-Trp-Phe-Trp-Leu-Leu-NH2 (9) and Tyr-D- Trp-Phe-D-Trp-Leu-Leu-NH2 (10)] were synthetized, and their ability to reverse multidrug resistance (MDR) was studied. Peptide derivatives 1, 4 and 5, with D-Trp or D-Tyr residues in the N-terminal position caused a marked inhibition of MDR in cancer cells. These MDR inhibitor compounds and epirubicin were demonstrated to have additive and synergistic antiproliferative effects in checkerboard experiments on human MDR1 gene-transfected mouse lymphoma cells in vitro. It is suggested that the MDR reversal effects of these anticancer peptide derivatives, together with their antiproliferative effects on lung cancer cells, may open up new horizons in cancer chemotherapy.

High mobility group box 1 protein induction by Mycobacterium bovis BCG.

UNLABELLED: High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 +/- 44 ng/mL) than that of LPS (84 +/- 12 ng/mL) or Staphylococcus aureus (150 +/- 14 ng/mL). BCG and Phorbol -12-myristate -13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 +/- 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. CONCLUSION: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.

Chronic infections and genetic factors in the development of ischemic stroke.

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.

The role of histamine in the intracellular survival of Mycobacterium bovis BCG.

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.