Mouse Foot Research Articles

Development of a Mouse Food Pad Model for Detection of Sub Clinical Leprosy

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.

Photodynamic therapy augments the efficacy of oncolytic vaccinia virus against primary and metastatic tumours in mice

Background:Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice.Methods:The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay.Results:Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue.Conclusion:These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV.

The LIM‐homeodomain transcription factor LMX1B supports the maintenance of differentiated podocytes by modulating the actin cytoskeleton

Mutations in the LMX1B gene cause a rare autosomal‐dominant disorder affecting the development of the limbs, eyes, brain and kidneys, called nail‐patella syndrome. In order to determine the role of LMX1B in the differentiation and maintenance of the renal filtration barrier we generated inducible podocyte‐specific Lmx1b knockout mice. Already one week after inactivation of Lmx1b in adult mice foot process effacement and rising proteinuria were detectable underlining the physiological significance of LMX1B in the maintenance of differentiated podocytes.Using a variety of different biophysical assays such as electric cell‐substrate impedance sensing, nano‐scale particle tracking, magnetic tweezer measurements and adhesion assays we were able to demonstrate that LMX1B affects the organization of the actin cytoskeleton. These findings were corroborated by micro array data which reveal LMX1B‐regulated genes encoding actin cytoskeleton‐associated proteins. Our data highlights the importance of LMX1B in fully differentiated podocytes and describes an essential role of LMX1B for the maintenance of an appropriately structured actin cytoskeleton in podocytes. This work was supported by the German Research Council through SFB 699.

Inhibition of Inducible Nitric Oxide Synthase Attenuates Monosodium Urate-induced Inflammation in Mice

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor N6-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-α, and IL-1β were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

Anti‐Inflammatory Potential of Ethanolic Leaf Extract of Eupatorium adenophorum Spreng. Through Alteration in Production of TNF‐α, ROS and Expression of Certain Genes

Search for a novel anti-inflammatory agent from a herbal source, such as Eupatorium adenophorum Spreng., a plant from the Eastern Himalayas, is of prime interest in the present investigation. Inflammation causes tissue destruction and development of diseases such as asthma, rheumatoid arthritis, and so forth. The ethanolic leaf extract of E. adenophorum (EEA) was administered intravenously and in other cases topically at the site of delayed type hypersensitivity (DTH) reaction in mouse foot paw induced with dinitrofluorobenzene. EEA can effectively inhibit DTH reaction and bring back normalcy to the paw much earlier than the controls. Efficacy of EEA on regulatory mechanisms for inflammation has also been considered. Intravenous administration of EEA increased the number of CD4+ T cells in spleen and tumor necrosis factor (TNF)-α in serum of DTH mice. Initially it was difficult to reconcile with the anti-inflammatory role of EEA and simultaneous induction of TNF-α, an established pro-inflammatory cytokine. EEA induces higher expression of TNF-α gene and amount of the cytokine in serum. We discussed the other role of TNF-α, its involvement in repairing tissue damage incurred in course of inflammatory reaction. EEA also induces TGF-β encoding a cytokine involved in tissue repair mechanism. EEA inhibits expression of another pro-inflammatory cytokine gene IL-1β and downregulates cycloxygenase 2 (COX2) gene responsible for metabolism of inflammatory mediators like prostaglandins. Furthermore, anti-inflammatory role of EEA is also revealed through its inhibition of hydroxyl radical generation. Notably EEA does not necessarily affect the expression of other inflammation-related genes such as IL-6, IL-10 and IKK. The present study reports and analyzes for the first time the anti-inflammatory property of the leaf extract of E. adenophorum.

Anti-inflammatory effects of peptide fragments of H2A histone and Oryza Sativa Japonica protein

Anti-inflammatory drugs are often of limited use due to low efficacy and toxic effects. The present study describes the anti-inflammatory effects of a novel nonapeptide termed IIIM1, using the mouse hind paw edema as an experimental model of inflammation. Multiple prophylactic injections of IIIM1 resulted in a significant reduction in carrageenan-induced foot pad swelling, both in mice and rats. A single prophylactic treatment of the peptide caused the maximal effect at 7–9 days between the initial peptide treatment and the subsequent carrageenan injection. A reduced inflammatory reaction was observed in transgenic mice constitutively expressing the peptide. A marked decrease in oxidative burst was observed in activated peritoneal macrophages obtained from peptide-treated mice. Furthermore, the sera of IIIM1-treated mice caused a significant decrease in the oxidative burst of macrophages. In addition, the reduction of hind paw swelling in mice injected with the sera of IIIM1-treated mice strongly suggests the presence of a circulating inducible factor responsible for the anti-inflammatory effect of the peptide. Previous LC/MS/MS analysis revealed the presence of a new peptide, termed RA1, in the sera of IIIM1-treated mice. RA1 was identified as a fragment of the Oryza Sativa Japonica protein. The anti-inflammatory effect of RA1 as evidenced by the reduction in carrageenan-induced hind paw swelling corresponded with the decrease in the oxidative burst of macrophages treated in vitro with this peptide. In conclusion, both IIIM1 and RA1 represent potential agents for the efficient treatment of inflammatory diseases that are currently incurable using presently available drugs.

Adjuvant effect of Cliptox™ on the protective immune response induced by an inactivated vaccine against foot and mouth disease virus in mice

Foot and Mouth Disease (FMD) is an acute disease caused by Foot and Mouth Disease Virus (FMDV) which causes important economy losses, this is why it is necessary to obtain a vaccine that stimulates a rapid and long-lasting protective immune response. Cliptox™ is a mineral microparticle that in earlier studies has shown adjuvant activity against different antigens. In this study we have examined the effects of Cliptox™ on the magnitude and type of immunity elicited in response to inactivated FMDV (iFMDV) vaccine. It was demonstrated that iFMDV-Cliptox ™ stimulates a specific antibody response detected in mucosal and in sera. The different isotype profiles elicited by inoculation with this vaccine indicate a Th1/Th2 response. Also, an increase in dendritic cells and macrophages in the spleen in comparison with the iFMDV vaccine iFMDV-Cliptox™ was detected. The Cliptox-iFMDV formulation was non toxic by using egg embryos and yielded increased protection against challenge with FMDV in the murine model. Our results show that the incorporation of Cliptox™ into FMDVi vaccine induces an increase of the specific protective immune response in mice and clearly indicate that Cliptox TM exert an (important) up-regulation on DC and MΦ. Additionally, Cliptox TM adjuvant can be used in vaccines for induction of mucosal immune response.

The effect of corticosteroids usage on bacterial killing, clearance and nerve damage in leprosy; Part 3 – Study of two comparable groups of 100 multibacillary (MB) patients each, treated with MDT + steroids vs MDT alone, assessed at 6 months post – release from 12 months MDT

To investigate effects of therapeutic usage of corticosteroids on M. leprae killing and clearance, on clearance of granuloma and on nerve damage in multibacillary (MB) leprosy patients. From a cohort of 400 untreated MB patients, a comparable group of 100 each receiving MDT + steroids (group A) vs MDT alone (group B) were assessed at 18 months as compared to month zero with respect to clinical and granuloma regression, M. leprae killing and clearance, and nerve functions. Analysis was performed using SPSS version 10.0. The significance of association was tested using Chi square and Fisher's exact tests. Regression of lesions assessed clinically and by histopathology was seen in 52% and 53% patients in group A and 46% and 63% in B respectively (P not significant). Clearance of bacteria assessed by bacteriological index (BI) in slit skin smears (SSS) and extent and intensity of antigen using anti-BCG staining were also comparable in the two groups. Multiplication of M. leprae in the mouse foot pad (MFP) indicating the presence of viable bacilli was seen in 14% and 16% of SSS positive BL-LLs patients in groups A and B respectively (P not significant). The occurrence of viable M. leprae was higher among patients with repeat reaction (19%) than single (11%). Using clinical tests (nerve palpation, monofilament and voluntary muscle testing), the proportion of sensory and motor nerves showing improvement or deterioration were similar in the two groups. However using nerve conduction studies, the overall proportion of nerves showing deterioration (22%) was significantly higher than improvement (9%) (P < 0.001). Treatment with MDT + corticosteroids does not adversely affect the clearance of granuloma, M. leprae and/or its antigens and M. leprae killing. However the continued presence of viable bacteria in > 14% of BL-LLs patients indicate that 12 months of MDT may be insufficient for complete bacterial killing. In both groups nerve conduction studies indicated that deterioration of nerves was high suggesting, MDT + corticosteroids was not very efficacious in the prevention or reversal of nerve damage. A better immuno-modulatory drug or a modified corticosteroid regime is needed.

Time to fatigue is increased in mouse muscle at 37°C; the role of iron and reactive oxygen species

Studies exploring the rate of fatigue in isolated muscle at 37 degrees C have produced mixed results. In the present study, muscle fibre bundles from the mouse foot were used to study the effect of temperature on the rate of muscle fatigue. Provided iron was excluded from the solutions, time to fatigue at 37 degrees C was increased compared to 22 degrees C (125 +/- 8% of 22 degrees C fatigue time). In contrast, when iron was present (approximately 1 microM), fatigue was accelerated (68 +/- 10%). Iron can increase reactive oxygen species (ROS), which are believed to accelerate fatigue. The addition of 25-100 microM H(2)O(2) at 22 degrees C reduced time to fatigue to 80-20% of the control, respectively. Iron was added to cultured primary skeletal muscle cells to determine if iron could increase ROS production. Neither iron entry nor ROS production were detected in non-contracting muscle cells. The addition of 8-hydroxyquinoline, which facilitates iron entry, to iron-ascorbic acid solutions caused a rapid rise in intracellular iron and ROS. Our results indicate that time to fatigue in vitro is increased at 37 degrees C relative to 22 degrees C, but the addition of ROS can accelerate fatigue. An increase in muscle iron can accelerate ROS production, which may be important during or following exercise and in haemochromatosis, disuse atrophy and sarcopenia.

The contemporary relevance of the mouse foot pad model for cultivating M. leprae

The contemporary relevance of the mouse foot pad model for cultivating M. leprae

Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L6)

In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells, a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L6) was established and the effect of curcumin on biological behavior of Hca-P/L6 was observed. Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential (Hca-P) was subcutaneously inoculated into the medioventral line of a mouse 615 and the first generation of metastatic tumor cells of inguinal lymph node (Hca-P/L1) was obtained. Then, Hca-P/L1 was screened by the route of mouse foot pad subcutaneously → lymph node → scale-up culture in vitro → mouse foot pad subcutaneously for five times consecutively. The sensitivity of two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L6) and two anchorage-dependent human hepatocarcinoma cell lines (SMC7721 and HepG2) to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after these cells had been pretreated by curcumin at the concentration of 15–240 μmol/L for 48 h. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 μmol/L in vitro, the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted microscope, cell growth curve and cell population doubling time; the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied by flow cytometry (FCM). The results showed Hca-P/L6 spreading to the lymph nodes at multiple sites in mice was screened from Hca-P. The lymph node metastatic rate was 100%. Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner (P<0. 05). At concentrations of 30–120 μmol/L, curcumin had more inhibition on murine ascites hepatocarcinoma cell lines than on human anchorage-dependent hepatocarcinoma cell lines. At concentrations of 60–240 μmol/L, curcumin had more inhibition on Hca-P/L6 with the 50% inhibitory concentration (IC50) of 51.48 μmol/L than on Hca-P with IC50 of 90.87 μmol/L. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days, the proliferations of Hca-P/L6 and Hca-P were inhibited (P<0.05) in a time-dependent manner (P<0.01) and the population doubling time of Hca-P/L6 and Hca-P was prolonged (P<0.01), and curcumin had more inhibition on Hca-P/L6 than on Hca-P (P<0.05). After pretreatment by 15 μmol/L curcumin for 48 h, the morphous of Hca-P/L6 was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G2/M phases. Hca-P/L6 was validated to be more sensitive to curcumin than Hca-P. Hca-P/L6 is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.

Alternative site of implantation affects tumor malignancy and metastatic potential in mice: Its comparison to the flank model

MC-C fibrosarcoma and B16F0 melanoma tumors were implanted intradermally in the dorsal region of the foot of mice. Tumor progression was compared to standard implantation in the flank. Although foot tumors only reached 13% (MC-C) and 25% (B16F0) of the mean volume of flank tumors, a more malignant phenotype in terms of histology and survival rate was observed in this type of tumors. Moreover, lung metastases were only detected in hosts bearing foot tumors, in contrast to MC-C and B16F0 populations with tumors growing in the flank. In addition, cellular influx and local immune reaction were higher in the dorsal region of the foot. According to our results, the dermis of the flank allows excessive tumor growth due to its low reactivity. Thus, differences in innate and adaptive immune effectors between the evaluated tumor microenvironments would account for the differences in tumor malignancy. Due to its striking differences with the standard flank inoculation, the tumor implantation model herein introduced could be a valuable tool to study the metastatic potential of different cell lines and the microenvironment components affecting tumor growth.

The Vascular Disrupting Agent 5,6‐Dimethylxanthenone‐4‐Acetic Acid Improves the Antitumor Efficacy and Shortens Treatment Time Associated with Photochlor‐sensitized Photodynamic Therapy In Vivo

In this report, we examined the antitumor activity of photodynamic therapy (PDT) in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a vascular disrupting agent currently undergoing clinical evaluation. BALB/c mice bearing subcutaneous CT-26 colon carcinomas were treated with PDT using the second-generation chlorin-based sensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (Photochlor) with or without DMXAA. Long-term (60-days) treatment outcome, induction of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), vascular damage (microvessel density, MVD) were evaluated as endpoints. In addition, treatment selectivity was evaluated using magnetic resonance imaging (MRI) and the foot response assay. A highly synergistic interaction was observed with the combination of low-dose DMXAA and PDT (48 J cm(-2) at 112 mW cm(-2)) resulting in approximately 60% long-term cures. The duration of the PDT session for this combination therapy protocol was only 7 min, while the duration of a monotherapy PDT session, selected to yield the equivalent cure rate, was 152 min. MRI showed markedly less peritumoral edema after DMXAA + short-duration PDT compared with long-duration PDT monotherapy. Similarly, DMXAA + PDT caused significantly less phototoxicity to normal mouse foot tissue than PDT alone. Increased induction of cytokines TNF-alpha and IL-6 (P < 0.001) was observed at 4 h followed by extensive vascular damage, demonstrated by a significant reduction in MVD at 24 h after combination treatment. In conclusion, Photochlor-sensitized PDT in combination with DMXAA exhibits superior efficacy and improved selectivity with clinically feasible illumination schemes. Clinical evaluation of this novel combination strategy is currently being planned.

Involvement of sphingosine-1-phosphate and S1P 1 in angiogenesis: Analyses using a new S1P 1 antagonist of non-sphingosine-1-phosphate analog

Chemical lead 2 (CL2) is the first non-sphingosine-1-phosphate (Sph-1-P) analog type antagonist of endothelial differentiation gene-1 (Edg-1/S1P 1), which is a member of the Sph-1-P receptor family. CL2 inhibits [ 3H]Sph-1-P/S1P 1 binding and shows concentration-dependent inhibition activity against both intracellular cAMP concentration decrease and cell invasion induced by the Sph-1-P/S1P 1 pathway. It also inhibits normal tube formation in an angiogenesis culture model, indicating that CL2 has anti-angiogenesis activity. This compound improved the disease conditions in two angiogenic models in vivo. It significantly inhibited angiogenesis induced by vascular endothelial growth factor in a rabbit cornea model as well as the swelling of mouse feet in an anti-type II collagen antibody-induced arthritis model. These results indicate that the Sph-1-P/S1P 1 pathway would have an important role in disease-related angiogenesis, especially in the processes of migration/invasion and tube formation. In addition, CL2 would be a powerful tool for the pharmacological study of the mechanisms of the Sph-1-P/S1P 1 pathway in rheumatoid arthritis, diabetes retinopathy, and solid tumor growth processes.

A Brazilian female with red brown nodules and plaques on her arms and chest

A 54-year-old Brazilian female presented with a 4-year history of subcutaneous nodules that began on the legs and later spread to involve the arms and chest. The patient noted occasional mild pruritus and burning of the lesions in addition to an 18-month complaint of night sweats and weight loss. She had immigrated to the United States 7 years previously. The physical examination revealed multiple, diffusely distributed, indurated, pink-brown nodules and plaques involving the upper extremities (Figs 6 and 7), chest, abdomen, and lower extremities. Two 4.0-mm punch biopsies were obtained from representative lesions on the right posterior arm, one for tissue culture and the other microscopic evaluation (Fig 8 and 9). 11.What is the most likely diagnosis? a.Palisading neutrophillic granulomatous dermatosis b.Lepromatous leprosy c.Tuberculoid leprosy d.Sarcoid e.Cutaneous leishmaniasis 12.Which of the following is false regarding Mycobacterium leprae? a.It is highly virulent b.It is an obligate intracellular parasite c.It is highly infective d.It favors cooler areas of the body e.It can replicate in mouse foot pads 13.Other findings may include which of the following? a.Lesional hypoaethesia b.Lesional anhidrosis c.“Glove and stocking” sensory deficits d.Loss of the lateral eyebrows e.c and d are both correct 14.Which of the following would you expect to be elevated? a.Interleukin-1 (IL-1) b.Tumor necrosis factor–alfa c.IL-2 d.IL-4 e.Interferon-gamma 15.Please select the appropriate stain for the causative organism. a.Wade–Fite b.Fontana–Masson c.Perl's stain d.Brown–Brenn e.Silver stain 16.The appropriate therapy would include which of the following? a.Dapsone b.Dapsone, rifampin, and clofazamine c.Minocycline d.Isoniazid, rifampin, and pyrazinamide e.Trimethoprim-sulfamethoxazole View Large Image Figure Viewer Download Hi-res image View Large Image Figure Viewer Download Hi-res image View Large Image Figure Viewer Download Hi-res image

Isolation and characterization of “Reprotoxin”, a novel protein complex from Daboia russelii snake venom

In snake venoms, non-covalent protein–protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA 2 (PLA 2), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6 kDa complex exhibits only phospholipase A 2 activity. In the presence of 8 M urea it is well resolved into protease (29.1 kDa), PLA 2 (13 kDa), and trypsin inhibitor (6.5 kDa) peaks. The complex showed an LD 50 of 5.06 mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3 mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named “Reprotoxin”. This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.

Enhanced local tumour control after single or fractionated radiation treatment using the hypoxic cell radiosensitizer doranidazole

Objective This study was designed to assess the potential of the nitroaromatic radiosensitizer doranidazole to preferentially enhance radiation-induced local control in a murine tumour. Methods A C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was used and treated when at 200 mm 3 in size. Doranidazole was dissolved in saline and injected intravenously. Radiation (240 kV X-rays) was locally administered to the tumours or normal feet of restrained non-anaesthetised animals. Response endpoints were local tumour control at 90 days and moist desquamation in foot skin 11–23 days after irradiation. Following logit analysis of the radiation dose–response curves the TCD50 (tumour) or MDD50 (skin) doses (radiation doses producing a response in 50% of treated mice) were estimated and a sensitizer enhancement ratio (SER; ratio of the TCD50 or MDD50 for radiation alone and radiation with drug) calculated. Statistical analysis was performed using a χ 2 test ( p < 0.05). Results The TCD50 value (±95% confidence interval) for radiation alone as a single treatment was 53 Gy (51–55). Injecting doranidazole (200 mg/kg) at 0, 30 or 60 min prior to irradiation significantly enhanced radiation response with the greatest effect seen at the 30-min interval [TCD50 = 40 Gy (37–44); SER = 1.3]. No enhancement occurred when the drug was given after radiation. Injecting different drug doses 30 min prior to irradiation showed a dose–response relationship; the respective SERs were 1.1, 1.3 and 1.8 at 50, 200 and 500 mg/kg. In skin, using the 200 mg/kg dose and a 30-min interval, the SER was only 1.1. Combining doranidazole and radiation in a fractionated schedule gave a tumour SER of 1.1. Conclusions Non-toxic doses of doranidazole significantly enhanced tumour response to single radiation treatments, an effect that was greater than that seen in a normal tissue. It also enhanced radiation given in a fractionated schedule. These effects were similar to those found with misonidazole and nimorazole, nitroaromatic radiosensitizers with clinical efficacy.

Relapses vs. reactions in multibacillary leprosy: proposal of new relapse criteria

To compare a new scoring system for multibacillary (MB) leprosy relapses, which combines time factor, risk factors and clinical presentation at relapse, to WHO criteria. Data were collected on all relapses diagnosed between 1998 and 2004 at the Marie-Adelaide-Centre in Karachi, Pakistan, including case histories, clinical manifestations, follow-up, bacterial indices, treatment and contacts. For the diagnosis of MB relapses a simple scoring system was developed and validated on a data-set of mouse foot pads (MFP)-confirmed relapses (Leprosy Reviews, 76, 2005, 241). Its sensitivity was further evaluated in the Karachi relapse cohort. The P-value was calculated with McNemar's test with continuity correction. The new scoring system that combines time factor, risk factors and clinical presentation at relapse had a higher sensitivity in MFP-confirmed relapses than the WHO-criteria (95%vs. 65%, P < 0.01). The sensitivity of the scoring system was also significantly higher than the WHO criteria in the 57 cases of MB-relapses diagnosed in Karachi (72%vs. 54%, P < 0.05). This new simple scoring system for diagnosing MB-relapses in leprosy should be further validated in a prospective study to confirm its superior sensitivity and to evaluate the specificity of these criteria by using MFP-confirmation for patients presenting with signs of activity after treatment.

The use of transgenic fluorescent mouse strains, fluorescent protein coding vectors, and innovative imaging techniques in the life sciences

paper by Fujiki et al. entitled ‘‘Quantification of greenfluorescence protein by in vivo imaging, PCR and flow cyto-metry: comparison of transgenic strains and relevance for fetalcell microchimerism’’ has a very promising approach to imagefetal cell microchimerism (1). The paper compares two typesof transgenic mice expressing green fluorescent protein (GFP)for the future study of fetal microchimerism—the processwhere fetal cells enter the maternal circulation, and in humanscan persist in the maternal blood and tissues for decades. TheGFP transgenic mice chosen for study are the C51B6/6-Tg(ACTB-EGFP) Osb/J (CAG) and the ROSA 26-EGFP. Theauthors conclude that the CAG mice have much brighter cells,but the ROSA 26-EGFP cells more consistently express GFP.The authors compare imaging, PCR, and flow cytometry(FCM) to image cells from these mice and conclude that flowcytometry is the method of choice, since ‘‘. . . due to the exist-ing status of this imaging technology and the purely quantita-tive nature of PCR amplification, FCM provides the best over-all approach for the quantitative and qualitative evaluation ofrare transgenic microchimeric fetal cells.’’The authors, however, are apparently unaware of thepower of fluorescence protein-imaging in vivo. Chishima et al.(2) pioneered this area and showed that single cancer cellsexpressing GFP could be readily imaged in autopsied miceagainst the large background of normal tissue. Subsequently,Yang et al. (3) showed that tumors could be whole-bodyimaged with GFP in live unrestrained mice. Yang et al. (4)then made two more important advances, and showed in skinflaps in live mice that single GFP cells could be imaged inmany organs. Yang et al. (5) then showed that tumor cellsexpressing red fluorescent protein (RFP) in the cytoplasm andGFP in the nucleus, as well as GFP host stromal cells, couldnoninvasively be imaged when grown in the mouse foot pad.Yamamoto et al. (6) and Yamauchi et al. (7,8) showedthat single cells labeled in the nucleus with GFP and in thecytoplasm with RFP could be imaged in mice both noninva-sively as well as in skin flaps. Yamauchi et al. (7,8) imaged thedual color cells in blood vessels as they trafficked in real time.Imaging cell trafficking was greatly enhanced by use of thevariable-magnification Olympus OV100 imaging system.Hayashi et al. (9) used the dual-colored cells and OlympusOV100 to image cells trafficking in lymphatics.Use of a high-powered variable-magnification imagingsystem, such as the OV100, rather than the IVIS 200 photoncounting device used by Fujiki et al. which does not produceimages, enables single fluorescent-protein-labeled cells to bevisualized in real time even during trafficking (10–12). Suchan approach offers many advantages over the IVIS 200 andflow cytometry, since cells can be visualized in the live animalwhich is very important for studying microchimerism (13).In parallel to the rapid development of instrumentation,many efforts have been made to optimize the existing ‘‘classical’’

Atividade antiinflamatória de carboidrato produzido por fermentação aquosa de grãos de quefir

Kefir, a symbiont microorganism suspension, presents benefic effects to health. Some kefir grains were cultivated in brown sugar, allowing to isolate a substance named CSQ. This was evaluated on a biologic essay of mouse foot edema, presenting an inhibitory activity of 30+4 % against carrageenan after the stimulus. It was observed that a cultivation mean containing sucrose, and not the milky mean, lead to the production of different sugar polymeric chains of kefir. The results in vivo suggest that the CSQ exerted an anti-inflammatory activity.