Effects of vitamin C on pathology and caspase-3 activity of kidneys with subacute endosulfan toxicity.

Endosulfan is an insecticide that is composed of two stereoisomers: α- and ß- endosulfan in an approximate ratio of 70:30. Owing to its widespread use, poisoning of both humans and animals is possible. We examined the toxic effects of endosulfan on New Zealand white rabbit kidneys. Rabbit kidneys were examined histopathologically and caspase-3 activity was detected using immunohistochemistry. Animals were divided into four groups: Group 1 was given a sublethal dose of endosulfan in corn oil by oral gavage daily for 6 weeks, Group 2 was given endosulfan + vitamin C during the same period, Group 3 was given corn oil daily and vitamin C on alternate days, Group 4 was given only corn oil daily throughout the experiment. By the end of experimental period, the concentration of α-endosulfan was greater than the ß-endosulfan concentration in the kidneys of both of endosulfan treated groups (Groups 1 and 2). Decreased accumulation of α- and ß-endosulfan was observed in Group 2, possibly because of the antioxidant effect of the vitamin C. Histopathological examination revealed hemorrhages, tubule cell necrosis, glomerular infiltration, glomerulosclerosis and proteinaceous material in the tubules, and Bowman spaces in the kidneys of Group 1. Caspase-3 reaction was stronger in Group 1 than in the other groups. Apoptotic activity was most frequent in proximal tubule cells. Endosulfan is toxic to rabbit kidneys. Vitamin C treatment reduced the accumulation of endosulfan in kidneys and reduced its toxicity.

Determination of sulphonamide residues in cattle meats by the Charm-II system and validation with high performance liquid chromatography with fluorescence detection.

The purpose of this study was determination of sulphonamide residues (sulphanilamide, sulphadiazine, sulphathiazole, sulphamerazine, sulphamethazine, sulphamethoxazole, and sulphadimethoxine) in cattle meat by the Charm II technique and the validation of sulphonamide levels by high performance liquid chromatography with fluorescence detector (HPLC-FLD). Of 157 meat samples, 9 samples (5.73%) were found positive by the Charm II method. To make quantitative confirmation of sulphonamide content of positive samples, HPLC-FLD was used and four samples were confirmed as positive. In HPLC analysis, the limit of detection (LOD) was in the range 8-15 µg/kg and the limit of quantification (LOQ) was 13-25 µg/kg. Average recoveries of sulphonamides ranged from 44.6% to 81% with relative standard deviations below 6% (n=6). In conclusion, we consider that the results obtained in field screening by only using the Charm II system, as is common practice in Turkey and worldwide, are inadequate and thus the results should be confirmed by sensitive systems like HPLC.

Cadmium and lead contamination in vegetables collected from industrial, traffic and rural areas in Bursa Province, Turkey.

Cadmium and lead contamination of vegetables produced in rural areas of Bursa Province, Turkey, was found to be less contaminated than vegetables grown close to heavy traffic and industrial activities. The highest levels of cadmium and lead were found in lettuce; the lowest levels in vegetables were found in leeks. The lead levels in spinach grown in traffic areas were at least twofold higher than those found in industrial areas. For other vegetables, the results from industrial and traffic areas were almost identical. Lettuce grown in traffic areas had the highest amount of cadmium (0.81 +/- 0.25 mg kg(-1)) and lead (1.59 +/- 0.45 mg kg(-1)), whilst leeks grown in rural areas had the lowest levels of lead (0.10 +/- 0.03 mg kg(-1)) and cadmium (0.05 +/- 0.01 mg kg(-1)) compared with other vegetables. This study shows that people and animals living in the same area in Bursa are always exposed to metallic pollution and in turn the consumption of contaminated vegetables could lead to increased dietary intake.

Nasal respiratory support in premature infants: short-term physiological effects and comfort assessment.

AIM: To evaluate the effects of nasal respiratory support on physiologic parameters and comfort of premature infants, when compared to spontaneous breathing without nasal respiratory support. METHODS: This was a prospective, randomized, controlled, cross-over clinical study. Infants were enrolled into the study when in 'stable' condition (when discontinuation of nasal respiratory support was considered appropriate). Infants were randomized to receive first 3 h of nasal respiratory support (nasal continuous positive airway pressure or nasal intermittent mandatory ventilation) or to spontaneous breathing, and then were crossed-over to the other assignment. Each infant served as his own control. RESULTS: Fifty-four infants were included in the study (birth-weight: 1528 +/- 545 g; gestational age: 30.5 +/- 2.7 weeks). Average values of systolic, diastolic and mean blood pressure and discomfort score were significantly higher while respiratory rate was significantly slower on nasal respiratory support compared to spontaneous breathing. Heart rate was comparable on both modes. CONCLUSIONS: Nasal respiratory support in 'stable' premature infants is associated with increased blood pressure and increased discomfort, despite a decreased respiratory rate. The clinical importance of these effects is modest. Medical teams should consider these effects and balance its need with its adverse effects according to the clinical condition.

Tropomyosin-induced arthritis in rats.

OBJECTIVE: Immunization of rats with alpha-tropomyosin (TPM) led to arthritis, uveitis and dermatitis, typical features of Behçet's disease (BD). The present study characterizes the arthritic features of this animal model, not previously described. METHODS: Lewis rats were immunized with bovine alpha-TPM and another group of rats was treated with neutralizing anti- tumor necrosis factor-alpha (TNF-alpha) antibodies. RESULTS: Clinically more than 90% of the immunized rats developed severe acute arthritis 12 days after vaccination. Rats that were followed-up for 6 months had persistent inflammation of the leg joints. Histologic studies demonstrated predominant mononuclear infiltrations in the acute phase of arthritis; the chronic arthritic process resulted in cartilage and bone damage and abundant fibrosis which led to joint deformations. Male and female rats had a similar clinical course. Analysis of the splenocyte cytokine profile kinetics revealed a persistently high level of interferon-gamma (INF-gamma) and an increase in TNF-alpha secretion during the acute phase. Increasing levels of interleukin (IL)-10 heralded the decline in clinical arthritis. No IL-4 was detected. No arthritis was detected in the rats treated with anti-TNF-alpha antibodies. CONCLUSION: The data indicates that alpha-TPM serves as an autoantigen to induce acute and chronic destructive arthritis in rats. This model is a TNF-alpha dependent autoimmune disease, with a Th1 cytokine profile.

Hood versus mask nebulization in infants with evolving bronchopulmonary dysplasia in the neonatal intensive care unit.

OBJECTIVE: To compare infants' discomfort, nursing-time and caregiver preference, and assess the clinical efficiency (as a secondary outcome) of hood versus facemask nebulization in infants with evolving bronchopulmonary dysplasia (BPD) in the neonatal intensive care unit. STUDY DESIGN: A prospective, open, randomized, controlled crossover clinical trial. In total, 10 infants with BPD who were on inhaled beta-agonist bronchodilators and corticosteroids were randomly assigned to receive their nebulized treatments either by a facemask, or by a hood for 2-3 days, and then crossover to receive the same treatments with the other technique for another 2-3 days. Infants' discomfort, nursing-time, caregiver preference and clinical efficiency were compared. RESULTS: At baseline there was no significant clinical difference between the groups. Nurse-time required for administering the hood nebulization (mean+/-s.e.m.: 1.9+/-0.1 min) was significantly shorter than the time for mask nebulization (12.0+/-0.6 min, P<0.0001). Infants' discomfort score was significantly lower (0.1+/-0.04) for hood versus mask nebulization (2.5+/-0.2, P<0.0001). Nurses and parents unequivocally preferred the hood treatment. During both mask and hood nebulization therapies (2-3 days) clinical efficiency was comparable. While both methods caused an immediate (20 min post) clinical improvement, the immediate respiratory assessment change score was significantly greater for the hood versus the mask nebulization (0.62+/-0.27 versus 0.13+/-0.14, P<0.05). CONCLUSIONS: Nebulization of aerosolized medications in infants with evolving BPD by hood was less time-consuming for caregivers and was much better tolerated by the infants while being at least as effective as the conventional facemask nebulization.

Alpha tropomyosin as a self-antigen in patients with Behçet's disease.

We report for the first time a significant increased lymphoproliferative response to alpha tropomyosin as well as observing autoantibodies to tropomyosin observed in Behcet's disease (BD) patients with posterior uveitis. Peripheral blood mononuclear cells (PBMCs) from 18 BD patients with posterior uveitis, 18 patients with other forms of noninfectious uveitis, 9 patients with retinal damage due to photocoagulation as well as 18 healthy donors were evaluated for antigen-specific lymphoproliferative responses to alpha tropomyosin and its derivative peptides. The proliferative responses of PBMCs to these antigens were studied using (3)H thymidine incorporation assay. Serum samples were also screened by ELISA for autoantibodies against tropomyosin. Six of the 18 (33%) BD patients with posterior uveitis showed increased proliferative response to alpha tropomyosin or its derivative peptides, while none of the healthy, disease controls were positive. The mean lymphoproliferative responses to tropomyosin were significantly higher (P < 0.02) in the BD patients compared to healthy or disease controls. Higher titres of anti-tropomyosin antibodies were also seen in four of the 18 BD patients but none in the healthy or disease control groups (P < 0.002). The occurrence of these abnormalities supports a possible role for alpha tropomyosin as a self-antigen in a subset of patients with Behcet's disease.

Modulation of proteinase-K resistant prion protein by prion peptide immunization.

Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.

Self prion protein peptides are immunogenic in Lewis rats.

Prion diseases are caused by abnormal folding of the prion protein. The paradigm is that the prion protein is not immunogenic because the immune system must be tolerant to such a self protein. In an attempt to identify immunogenic prion peptides, we immunized Lewis rats with peptides that fitted the MHC class II RT1.B(1)motif. Both humoral and cellular immunity to the prion peptides were obtained without any harmful effects to young animals. However, when 8-month-old rats were immunized, a sixth (6/36) of the rats developed severe skin inflammation with concomitant hair loss. These findings suggest that immunity to self-prion peptides can be readily induced in Lewis rats and that this immune response may have pathogenic consequences in older rats.

Suppression of experimental autoimmune encephalomyelitis by intravenously administered polyclonal immunoglobulins.

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats either by active immunization with myelin basic protein (MBP) or by adoptive transfer using anti-MBP specific CD4(+)T cells. Treatment with human polyclonal immunoglobulins (IgG) effectively suppressed active EAE. Time-dependent experiments demonstrated that the effect of IgG was manifested only when treatment was given immediately after immunization; administration from day 7 after disease induction did not suppress the disease. In the adoptive transfer model of EAE, IgG had no effect in vivo. However, pretreatment in vitro of the antigen-specific T-cells with IgG inhibited their ability to mediate adoptive EAE, as it did in active EAE. Similarly, in vitro IgG pretreatment of the antigen-specific T-cells suppressed the proliferative response to MBP. Fluorescent Activated Cell Sorter (FACS) analysis demonstrated the binding of IgG to activated T-cell lines that was inhibited by soluble Fc molecules. The differential effects of IgG on active EAE and on the adoptive transfer of EAE suggest that IgG in vivo can suppress disease by acting during the early phase of the immune response which involves naive T cells. The inhibition of T-cell proliferation and adoptive transfer of EAE by incubation of T cells in vitro appears to require higher concentrations of IgG than those obtained in vivo.

Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion.

Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9+/-0.4, n = 25, compared with 8.3+/-0.4, n = 12; p<0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0+/-1; n = 6) than that observed in control rats treated with PBS (2.0+/-0.8; n = 6; p<0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1+/-0.8 (n = 6) compared with a score of 3.0+/-0.8 (n = 5; p<0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.

T cell immunity to copolymer 1 confers neuroprotection on the damaged optic nerve: possible therapy for optic neuropathies.

We recently reported that the posttraumatic spread of degeneration in the damaged optic nerve can be attenuated by the adoptive transfer of autoimmune T cells specific to myelin basic protein. However, it would be desirable to obtain immune neuroprotection free of any possible autoimmune disease. In an attempt to obtain disease-free immune neuroprotection, we used the synthetic four-amino acid polymer copolymer 1 (Cop-1), which is known not to be encephalitogenic despite its cross-reactivity with myelin basic protein. We show here that active immunization with Cop-1 administered in adjuvant, as well as adoptive transfer of T cells reactive to Cop-1, can inhibit the progression of secondary degeneration after crush injury of the rat optic nerve. These results have implications for the treatment of optic neuropathies.

Selection of anti-myelin basic protein T-cell lines in the lewis rat: V-beta 8.2 dominance and conserved complementarity-determining-region-3 motifs are dependent on serine at position 78 of myelin basic protein.

In the Lewis rat, the dominant T cell repertoire to myelin basic protein (MBP) is directed to the peptide 71-87 and the T cell receptors of pathogenic T cells are of the Vbeta 8.2 genotype with short CDR3 sequences having a characteristic motif. However, this paradigm has been reached through analysis of long-term encephalitogenic lines and clones. We initiated the present study to examine the process of selection of the TCR Vbeta 8.2 and characteristic CDR3 motifs upon immunization with guinea-pig MBP, and rat or guinea-pig 71-87 peptides. We found that the dominance of Vbeta 8.2 developed progressively over 4-6 in vitro stimulations. Following immunization with rat 70-86, which differs from the guinea-pig peptide in one amino acid at position 78, the dominance of Vbeta 8.2 and the characteristic CDR sequences are not seen. Thus, Vbeta 8.2 dominance and specific CDR3 TCR motifs are seen with heterologous GpMBP but not with self rat MBP.

Autoimmune T cells retard the loss of function in injured rat optic nerves.

We recently demonstrated that autoimmune T cells protect neurons from secondary degeneration after central nervous system (CNS) axotomy in rats. Here we show, using both morphological and electrophysiological analyses, that the neuroprotection is long-lasting and is manifested functionally. After partial crush injury of the rat optic nerve, systemic injection of autoimmune T cells specific to myelin basic protein significantly diminished the loss of retinal ganglion cells and conducting axons, and significantly retarded the loss of the visual response evoked by light stimulation. These results support our challenge to the traditional concept of autoimmunity as always harmful, and suggest that in certain situations T cell autoimmunity may actually be beneficial. It might be possible to employ T cell intervention to slow down functional loss in the injured CNS.

Autoimmune T cells as potential neuroprotective therapy for spinal cord injury.

Autoimmune T cells against central nervous system myelin associated peptide reduce the spread of damage and promote recovery in injured rat spinal cord, findings that might lead to neuroprotective cell therapy without risk of autoimmune disease.

Experimental autoimmune keratitis induced in rats by anti-cornea T-cell lines.

PURPOSE: Idiopathic inflammation of the cornea, keratitis, has been proposed to result from an autoimmune process, but thus far no convenient animal model of keratitis exists. An attempt was made to establish an animal model for keratitis, to investigate possible autoimmune mechanisms. METHODS: T-cell lines were established from lymph node cells removed from rats immunized with bovine corneal epithelium (BCE) extract. After restimulation in vitro with BCE or a specific corneal antigen, the cells were transferred by intraperitoneal injection into naive rats, rats subjected to total body irradiation, or rats in which only one eye was irradiated. RESULTS: Neither direct immunization with corneal antigens nor transfer of activated anti-corneal T-cells into naive rats gave any signs of keratitis. Irradiation alone did not induce corneal inflammation. Transfer of corneal-specific activated T cells into irradiated rats produced keratitis starting around day 4 and culminating around day 8. The disease was self-limiting and the severity dependent on the dose and site of radiation. Keratitis was characterized by corneal haze, conjunctival and episcleral hyperemia, episcleral hemorrhages, chemosis, corneal infiltrates, and vascularization. Immunohistochemistry showed T-cell and macrophage infiltration of epithelium and stroma in the affected corneas. CONCLUSIONS: Thus, keratitis may be produced by T cells reactive to corneal antigens, provided that the target tissue has been made susceptible by irradiation. The effectiveness of T-cell vaccination in preventing adoptive keratitis suggests that systemic as well as local tissue factors may regulate the disease process.

The 60-kDa heat shock protein modulates allograft rejection.

Allograft rejection is a process of immune reactivity triggered by foreign transplantation antigens. We now demonstrate that the 60-kDa heat shock protein (hsp60), a molecule that is identical in the donor and the recipient, can regulate allograft immunity. In wild-type mice, hsp60 expression was greatly enhanced in allografts being rejected. By using MHC class II (Ealpha) promoter hsp60 transgenic mice either as donors of skin with enhanced expression of hsp60, or as allograft recipients with decreased hsp60 autoimmunity, we found that augmented expression of mouse hsp60 in the allograft accelerated its rejection, whereas reduced autoimmunity to mouse hsp60 in graft recipients delayed the process. Moreover, in nontransgenic mice, therapeutic administration of hsp60 or hsp60 peptides, known to modulate naturally occurring hsp60 autoimmunity, led to delayed allograft rejection. Thus, we demonstrate that hsp60 expression and hsp60 autoimmunity can influence and modify the immune response to foreign antigens. Hence, autoimmunity to self-hsp60 epitopes is not necessarily an aberration, but may serve physiologically and therapeutically to modulate foreign immunity.

Non-coding plasmid DNA induces IFN-gamma in vivo and suppresses autoimmune encephalomyelitis.

Regulatory sequences used in plasmids for naked DNA vaccination can modulate cytokine production in vivo. We demonstrate here that injection of plasmid DNA can suppress the prototypic T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis, by inducing IFN-gamma.

Diversity of the B cell repertoire to myelin basic protein in rat strains susceptible and resistant to EAE.

Myelin basic protein (MBP) is a major protein of central nervous system myelin which can induce experimental autoimmune encephalomyelitis (EAE) in susceptible laboratory animals. The role of T cells in the induction of EAE has been extensively studied, but the antibody response to MBP has not been well characterized. In the present work, we immunized rats with encephalitogenic guinea-pig MBP and mapped autoreactive antibodies binding to peptides in the rat MBP sequence. We studied the responses of the Lewis rat strain, susceptible to EAE, and the responses of the Fischer and Brown-Norway (BN) rats, resistant to EAE. We found that Lewis rats immunized to guinea-pig MBP develop antibodies to a diversity of MBP epitopes with a dominance of MBP peptide p11-30 and peptides in the 71-140 region. Fischer rats showed a similar pattern of antibody specificities, but with higher titers than the Lewis rats. BN rats, in contrast, developed a very low titer of antibodies and lacked a response to p11-30. Thus, there is no clear correlation between the nature of the anti-MBP antibody response and the state of susceptibility or resistance to EAE induction in the different rat strains.

Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy.

Autoimmunity to antigens of the central nervous system is usually considered detrimental. T cells specific to a central nervous system self antigen, such as myelin basic protein, can indeed induce experimental autoimmune encephalomyelitis, but such T cells may nevertheless appear in the blood of healthy individuals. We show here that autoimmune T cells specific to myelin basic protein can protect injured central nervous system neurons from secondary degeneration. After a partial crush injury of the optic nerve, rats injected with activated anti-myelin basic protein T cells retained approximately 300% more retinal ganglion cells with functionally intact axons than did rats injected with activated T cells specific for other antigens. Electrophysiological analysis confirmed this finding and suggested that the neuroprotection could result from a transient reduction in energy requirements owing to a transient reduction in nerve activity. These findings indicate that T-cell autoimmunity in the central nervous system, under certain circumstances, can exert a beneficial effect by protecting injured neurons from the spread of damage.