Effects of Caffeine and Chlorogenic Acid on Nonalcoholic Steatohepatitis in Mice Induced by Choline-Deficient, L-Amino Acid-Defined, High-Fat Diet.

Several recent experimental studies have investigated the effects of caffeine and chlorogenic acid (CGA), representative ingredients of coffee, on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). However, the results are conflicting, and their effects are yet to be clarified. In the present study, we examined the effects of caffeine and CGA on choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice, relatively new model mice of NASH. Seven-week-old male C57BL/6J mice were divided into the following groups: Control diet (control), CDAHFD (CDAHFD), CDAHFD supplemented with 0.05% (w/w) caffeine (caffeine), and CDAHFD supplemented with 0.1% (w/w) CGA (CGA). After seven weeks, the mice were killed and serum biochemical, histopathological, and molecular analyses were performed. Serum alanine aminotransferase (ALT) levels were significantly higher in the caffeine and CGA groups than in the CDAHFD group. On image analysis, the prevalence of Oil red O-positive areas (reflecting steatosis) was significantly higher in the caffeine group than in the CDAHFD group, and that of CD45R-positive areas (reflecting lymphocytic infiltration) in the hepatic lobule was significantly higher in the caffeine and CGA groups than in the CDAHFD group. Hepatic expression of interleukin (IL)-6 mRNA was higher in the caffeine and CGA groups than in the CDAHFD group, and the difference was statistically significant for the caffeine group. In conclusion, in the present study, caffeine and CGA significantly worsened the markers of liver cell injury, inflammation, and/or steatosis in NASH lesions in mice.

The citrus flavanone naringenin attenuates zymosan-induced mouse joint inflammation: induction of Nrf2 expression in recruited CD45+ hematopoietic cells.

BACKGROUND: Naringenin is a biologically active analgesic, anti-inflammatory, and antioxidant flavonoid. Naringenin targets in inflammation-induced articular pain remain poorly explored. METHODS: The present study investigated the cellular and molecular mechanisms involved in the analgesic/anti-inflammatory effects of naringenin in zymosan-induced arthritis. Mice were pre-treated orally with naringenin (16.7-150 mg/kg), followed by intra-articular injection of zymosan. Articular mechanical hyperalgesia and oedema, leucocyte recruitment to synovial cavity, histopathology, expression/production of pro- and anti-inflammatory mediators and NFκB activation, inflammasome component expression, and oxidative stress were evaluated. RESULTS: Naringenin inhibited articular pain and oedema in a dose-dependent manner. The dose of 50 mg/kg inhibited leucocyte recruitment, histopathological alterations, NFκB activation, and NFκB-dependent pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-33), and preproET-1 mRNA expression, but increased anti-inflammatory IL-10. Naringenin also inhibited inflammasome upregulation (reduced Nlrp3, ASC, caspase-1, and pro-IL-1ß mRNA expression) and oxidative stress (reduced gp91phox mRNA expression and superoxide anion production, increased GSH levels, induced Nrf2 protein in CD45+ hematopoietic recruited cells, and induced Nrf2 and HO-1 mRNA expression). CONCLUSIONS: Naringenin presents analgesic and anti-inflammatory effects in zymosan-induced arthritis by targeting its main physiopathological mechanisms. These data highlight this flavonoid as an interesting therapeutic compound to treat joint inflammation, deserving additional pre-clinical and clinical studies.

Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells

BACKGROUND: Different methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells. MATERIAL AND METHODS: One hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed. RESULTS: Dental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ ml of thermolysine for 40 minutes and sonicating the culture for one minute. CONCLUSIONS: Dental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agents

Tumor-associated primo vascular system is derived from xenograft, not host.

The primo vascular system (PVS), which is composed of very small primo-vessels (PV) and primo-nodes (PN), has recently emerged as a third component of circulatory system. Here, we report the presence of a tumor derived PVS in murine xenografts of human histiocytic lymphoma (U937) in close proximity to the tumor. Within this system, PNs are small (~500-600 µM diameter) membranous sac-like structures which contain numerous small cells which can be demonstrated by DAPI staining. Hematoxylin and Eosin (H&E) staining of the peri-tumoral PVS shows the presence of loose structures lined by fibroblasts but filled with dense fibers, cells, lacunae and nerve-like structures. The origin and type of cells within the PVS was characterized by immunostaining with antibodies for CD68, CD45 and lysozyme. The results of these studies reveal that the PVS of the xenograft originates from the human U937 tumor cells. qRT-PCR analysis of mRNA isolated from PVS cells reveals a striking predominance of human, rather than mouse, sequences. Of particular interest, human stem cell specific transcription factors were overexpressed, most notably KLF4, an upstream regulator of NANOG which maintains the pluripotent and undifferentiated state of stem cells. These results suggest that the cells present within the PVS are derived from the human xenograft and suggests that the primo-vessels associated with the xenografted tumor may provide a safe haven for a select population of cancer stem cells. Further understanding of the biological properties of these cells may allow the development of new anti-cancer interventions.

Intravesical chondroitin sulfate inhibits recruitment of inflammatory cells in an acute acid damage "leaky bladder" model of cystitis.

OBJECTIVE: To investigate whether a physiologic effect of "glycosaminoglycan (GAG) replenishment therapy" altered recruitment of inflammatory cells in an acute bladder damage model. Replacement of the GAG layer with intravesically administered GAGs is an effective therapy for interstitial cystitis in at least some patients. Intravesically administered chondroitin sulfate was previously shown to bind to and restore the impermeability of surface-damaged ("leaky") urothelium to small ions. METHODS: Rat bladders were damaged with 10 mM HCl. Negative control bladders were treated with phosphate-buffered saline. On the following day, the animal bladders were treated with 20 mg/mL chondroitin sulfate in phosphate-buffered saline, and the negative and positive controls were treated with phosphate-buffered saline alone. At 2 and 4 days after treatment with chondroitin sulfate, the rats were killed, and sections of their bladders were analyzed using toluidine blue staining for mast cell immunohistochemical labeling using antibodies against CD45 for lymphocytes and myeloperoxidase for neutrophils. RESULTS: Chondroitin sulfate treatment reduced the recruitment, in a statistically significant manner, of inflammatory cells, including neutrophils and mast cells to the suburothelial space but did not alter recruitment of CD45-positive lymphocytes. CONCLUSION: For the first time, we have demonstrated that intravesical GAG replenishment therapy also produces a physiologic effect of decreasing recruitment of inflammatory cells in an acute model of the damaged bladder. These findings support the use of intravesically administered GAG for bladder disorders that result from a loss of impermeability, including interstitial, radiation, and chemical cystitis, and possibly others as well.

The effect of royal jelly on CD3(+), CD5(+), CD45(+) T-cell and CD68(+) cell distribution in the colon of rats with acetic acid-induced colitis.

BACKGROUND: Traditional medicines and health supplements have historically been used to treat many illnesses but most of them have not been evaluated objectively to prove their efficacy. We have been investigating the effects of royal jelly (RJ) supplements on acetic acid-induced colitis on the distribution of CD3(+), CD5(+), CD45(+) T-cell and CD68(+) cells in rats. METHODS: The rats were divided into four equal groups: control group, royal jelly-treated (RJ - 150mgkg(-1) body weight), acetic acid-treated (colitis) and acetic acid-treated (colitis)+royal jelly (CRJ - 150mgkg(-1) body weight). Colitis was induced by intracolonic instillation of 4% acetic acid; the control group received physiological saline (10mLkg(-1)). Colon samples were obtained under deep anaesthesia from animals in four groups. Tissues were fixed in 10% formalin neutral buffer solution for 24h and embedded in paraffin. RESULTS: The proliferative response of CD3(+) and CD45(+) T cells stimulated with colitis was affected by colitis treated with RJ. No differences were found in CD5(+) T cells and CD68(+) macrophages in the colitis treated with RJ. CONCLUSIONS: This study has shown that RJ has anti-inflammatory and cell regeneration effect in the colon of rats with acetic acid induced colitis.

Protein tyrosine phosphatase CD45 as a molecular biosensor of hydrogen peroxide generation in cell culture media.

We have designed a useful method of assessing reactive oxygen species generation in biological fluids. The novel assay utilizes tyrosine phosphatase CD45 as a biosensor of oxidative stress. Applying this new method, we examined oxygen species generation in the following cell culture media: RPMI 1640, DMEM, DMEM enriched with pyruvate and MEM. We discovered that the media (especially RPMI 1640) significantly reduced the activity of protein tyrosine phosphatase. The media-caused inactivation of CD45 was reversible after treatment with dithiothreitol being a powerful reducing agent. Interestingly, the media supplemented with catalase did not exhibit any inhibitory effect on CD45 activity which suggests a hydrogen peroxide-mediated mechanism of the enzyme inactivation. In addition to that, we assessed the impact of oxidative stress level on the activity of CD45 as measured in Jurkat cells cultured in RPMI 1640 either exposed or not exposed to the light of laminar flow cabinet fluorescent lamp. We found that Jurkat cells that were exposed to light displayed ca. 20% lower activity of CD45 than the cells protected against the light. The obtained results indicate that production of hydrogen peroxide in the medium leading to inhibition of CD45 was light-dependent, and that careful protection of cell culture media from the light may help to prevent the artifact in cell studies. Hydrogen peroxide, responsible for CD45 inactivation, can be generated in cell culture media after exposition to light due to photoreactive amino acids present in the media.

Quantitative in vivo and ex vivo confocal microscopy analysis of corneal cystine crystals in the Ctns knockout mouse.

PURPOSE: The purpose of this study was to assess the ability of quantitative in vivo confocal microscopy to characterize the natural history and detect changes in crystal volume in corneas from a novel animal model of cystinosis, the cystinosin (Ctns(-/-)) mouse. METHODS: Two Ctns(-/-) mice and one C57Bl/6 mouse were examined at each of the following time points: 2, 3, 5, 7, 10, 12, and 14 months of age. In vivo confocal microscopy scans were performed in 4 different regions of the cornea per eye. After, animals were sacrificed and cornea blocks evaluated for cell morphology using phalloidin and lymphocytic infiltration using CD45 antibodies by ex vivo confocal microscopy. Cystine crystal content in the cornea was measured by calculating the pixel intensity of the crystals divided by the stromal volume using Metamorph Image Processing Software. RESULTS: Corneal crystals were identified in Ctns(-/-) eyes beginning at 3 months of age and increased in density until 7-12 months, at which time animals begin to succumb to the disease and corneas become scarred and neovascularized. Older Ctns(-/-) mice (7 months and older) showed the presence of cell infiltrates that stained positively for CD45 associated with progressive keratocyte disruption. Finally, at 12 months of age, decreased cell density and endothelial distortion were detected. CONCLUSIONS: Confocal microscopy identified corneal crystals starting at 3 month old Ctns(-/-) eyes. Cystine crystals induce inflammatory and immune response with aging associated with loss of keratocyte and endothelial cells. These findings suggest that the Ctns(-/-) mouse can be used as a model for developing and evaluating potential alternative therapies for corneal cystinosis.

Pollen grains induce a rapid and biphasic eczematous immune response in atopic eczema patients.

INTRODUCTION: Eczematous reactions to type I allergy-inducing antigens are documented in a subgroup of patients with atopic eczema. Yet, the underlying immunological mechanisms are not well understood. MATERIAL AND METHODS: To delineate the effect of native pollen grains on human skin of healthy and atopic individuals we performed patch tests (atopy patch test with native pollen grains, PPT). Nickel patch tests (NPT) served as an established model of contact dermatitis. Skin site biopsies were taken 6-96 h after allergen application and investigated immunohistochemically. RESULTS: Histology of positive patch tests showed an influx of mononuclear cells (predominantly CD4+, CD25+, CD45RO+). This influx was detected earlier in the PPT reaction than in the immune response to nickel. A biphasic cytokine response could be detected in the PPT: IL-5 dominated in the early, IFN-gamma in the late phase. The NPT was continuously dominated by IFN-gamma. Dendritic cell subpopulations imitated the earlier kinetics of the mononuclear infiltrate. DISCUSSION: Thus, pollen grains induce eczematous reactions in susceptible individuals. This reaction appears clinically and immunohistochemically similar to the contact hypersensitivity reaction to nickel but follows a faster kinetic and a biphasic course: Th2 and IgE in the early (24 h) and Th1 predominance in the late (96 h) phase.

Activation-induced cell death signalling in CD4+ T cells by staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is a potent stimulator of CD4+ and CD8+ T cells, the immunotoxic action of which remains unclear. We investigated the in vitro effects of SEA on freshly isolated human peripheral blood lymphocytes depleted of CD8+ T cells. Proliferation and flow cytometry analysis indicated that SEA generated an activation-induced cell death (AICD) phenomenon that was characterized by an increased expression of the chemokine receptor CCR5 on the CD4+/CD45RO+ T cell subset. Incubation of cells with glycoprotein secretion inhibitor monensin A completely blocked cell proliferation, affecting mainly the CD4+/CD45RO+ T cell subset. The IL-2 mRNA levels were increased just hours after SEA stimulation, accompanied by an increase in the expression of CD25, indicating a possible involvement of IL-2 in the AICD process. We observed a 15-fold mRNA reduction of the transcription factor Yin Yang 1 (YY1) at the proliferation peak, and an increase of the receptors CCR5, CD95 and DR5 on the CD45RO+/CD4+ T cell subset. These findings suggest that SEA triggers a TCR-mediated AICD mechanism in CD4+ T cells, the intracellular signalling of which is probably modulated, at least, by YY1.

The immunosuppressive drug FK778 induces regulatory activity in stimulated human CD4+ CD25- T cells.

The induction of transplantation tolerance involves a T-cell-mediated process of immune regulation. In clinical transplantation, the use of immunosuppressive drugs that promote or facilitate this process would be highly desirable. Here, we investigated the tolerance-promoting potential of the immunosuppressive drug FK778, currently under development for clinical therapy. Using a human allogeneic in vitro model we showed that, upon T-cell receptor (TCR) triggering, FK778 induced a regulatory phenotype in CD4+ CD25- T cells. Purified CD4+ CD25- T cells primed in the presence of FK778 showed hyporesponsiveness upon restimulation with alloantigen in the absence of the drug. This anergic state was reversible by exogenous interleukin-2 (IL-2) and was induced independent of naturally occurring CD4+ CD25+ regulatory T cells. Pyrimidine restriction was a crucial requirement for the de novo induction of regulatory activity by FK778. The FK778-induced anergic cells showed suppressor activity in a cell-cell contact-dependent manner; were CD25(high), CD45RO+, CD27-, and CD62L-; and expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and FoxP3. The cells revealed delayed p27(kip1) degradation and enhanced phosphorylation of STAT3. In conclusion, the new drug FK778 shows tolerizing potential through the induction of a regulatory T-cell subset in CD4+ CD25- T cells.

Seasonal dynamics of chemokine receptors and CD62L in subjects with asymptomatic skin sensitization to birch and grass pollen.

BACKGROUND: Asymptomatic skin sensitization (AS) has been shown to be a risk factor for respiratory allergic disease. CCR4, CXCR1 and CD62L have all been assigned a role in the immunopathogenesis of allergy. Memory T-cell expression of CCR4, CXCR1 and CD62L has not hitherto been investigated in subjects with AS. METHODS: We investigated seasonal CD4 memory T-cell expression of the chemokine receptors CCR4, CXCR1 as well as L-selectin (CD62L) in fresh cultures derived from symptomatic atopics (SAs), subjects with AS and healthy controls (HCs). Peripheral blood mononuclear cells from all three groups were isolated during birch and grass pollination as well as in the following winter. CD4 memory T-cell expression of CCR4, CXCR1 and CD62L was determined by flow-cytometry. RESULTS: During spring and summer, a significantly increased proportion of memory T cells expressed CCR4, CXCR1 and CD62L in SAs when compared with subjects with AS and HCs. Only SAs exhibited seasonal fluctuations in numbers of CCR4, CXCR1 and CD62L positive memory T cells. CONCLUSION: Although clearly IgE sensitized, subjects with AS have significant diminished numbers of CCR4, CXCR1 and CD62L positive memory T cells, during pollination, when compared with SAs. In contrast to SAs, cultures derived from subjects with AS did not display seasonal variation. Our findings explain the lack of clinical symptoms, during pollination, in subjects with AS.

Randomized double-blinded and controlled clinical trial on treatment of HIV/AIDS by Zhongyan-4.

OBJECTIVE: To assess the efficacy and safety of Zhongyan-4 (ZY-4, a Chinese herbal preparation worked out according to the therapeutic principle of supplementing qi, nourishing Yin, clearing heat and detoxication) in treating HIV/AIDS patients in the early or middle stage. METHODS: Adopted was randomized double-blinded and placebo-parallel-controlled method, with 72 HIV/AIDS patients randomly divided into the ZY-4 group (36 patients) treated with ZY-4 and the control group (36 patients) treated with placebo. The treatment course was six months. The index of CD(4)(+), CD(8)(+) counts, body weight, clinical symptom scoring were estimated at 4 time points (0, 1, 3 and 6 month in the course), and also the viral load before and after treatment. The whole course of observation was completed in 63 patients, 30 in the ZY-4 group and 33 in the control group. RESULTS: CD(4)(+) count in the ZY-4 group got elevated by 7.70 +/- 150.96/mm(3) on average, while that in the control group lowered by 27.33 +/- 85.28/mm(3). Fifteen out of the 30 patients in the ZY-4 group had their CD(4)(+) count increased, which was evidently much higher than that in the control group (8/33, P < 0.05), suggesting that the efficacy of ZY-4 is superior to that of placebo in elevating CD(4)(+) count. Moreover, ZY-4 showed actions in elevating CD(45)RA(+) and CD(8)(+) count, reducing HIV virus load, improving clinical symptom/sign and increasing body weight of patients. No obvious adverse reaction was found in the clinical trial. CONCLUSION: ZY-4 has an immunity-protective and/or rebuilding function in HIV/AIDS patients in the early and middle stage, and also shows effects in lowering viral load, increasing body weight and improving symptoms and signs to a certain degree.

Immunological characteristics of subjects with asymptomatic skin sensitization to birch and grass pollen.

BACKGROUND: Asymptomatic skin sensitization (AS) has been shown to be a risk factor for respiratory allergic disease. OBJECTIVE: We investigated allergen and recall antigen-driven T cell proliferation, cytokine production and T cell expression of the chemokine receptor CCR4, in cultures derived from symptomatic atopics (SA), subjects with AS and healthy controls (HC). Numbers of allergen-specific precursor T cells in all three groups were also estimated. METHODS: Peripheral blood mononuclear cells from the three groups were isolated and stimulated with allergen and tetanus toxoid. Proliferation, cytokine production and CCR4 expression were measured by flow cytometry. RESULTS: A significantly increased proportion of CD4(+) memory T cells proliferated in response to allergen in SA as compared with subjects with AS (P<0.001) and HC (P<0.001). Only in SA was expansion of CD4(+)CCR4(+) T cells, after allergen stimulation observed. SA had higher frequencies of allergen-specific T cells than subjects with AS and HC (P=0.02, for both). With regard to allergen-induced production of T-helper type 1 (Th1) and Th2 cytokines, subjects with AS and HC resembled each other, while differing significantly from SA. CONCLUSION: We conclude, that subjects with AS, although clearly IgE sensitized, have significant diminished numbers of allergen-specific T cells as well as decreased allergen-induced CD4(+) memory T cell proliferation as compared with SA. To a large extent, our findings are capable of explaining the immunological characteristics associated with AS. Our findings may serve as better prognostic markers for subsequent allergic progression, than previously described clinical and paraclinical characteristics.

Intravascular large B-cell lymphoma involving hemangiomas: an unusual presentation of a rare neoplasm.

We report the clinicopathological features of two cases of intravascular large B-cell lymphoma involving cutaneous hemangiomas. The cases were identified from the consultation files of two of the authors. Both patients were women, 64 and 55 years of age, who presented with long-standing cutaneous hemangiomas of the posterior scalp and left shoulder, respectively. The lesions were brought to medical attention by an increase in size and change in color. Biopsies and immunohistochemical evaluation of the hemangiomas revealed extensive involvement by intravascular large B-cell lymphoma. The neoplastic cells were diffusely positive for CD20 in both cases and negative for CD3, pan-cytokeratin (AE1/AE3), epithelial membrane antigen, S-100, Factor VIII-related antigen, CD34 and CD31. Disease was limited to the hemangiomas in both patients. Treatment consisted of chemotherapy (both patients) and adjuvant radiation therapy (one patient). One patient had a recurrence of disease 33 months after initial diagnosis, leading to an autologous stem cell transplant. The other patient is without evidence of disease 27 months after initial diagnosis. Although this is a rare neoplasm, it is important to consider intravascular large B-cell lymphoma in the differential diagnosis of vascular lesions containing intravascular neoplastic cells.

Tazarotene 0.1% gel for refractory mycosis fungoides lesions: an open-label pilot study.

BACKGROUND: Topical skin-directed therapies are used to induce remissions in early-stage mycosis fungoides (MF). They are rarely curative, and responding patients are subject to frequent relapses, emphasizing the need for alternative therapies. OBJECTIVE: We sought to evaluate the efficacy and tolerability of topical tazarotene 0.1% gel as adjuvant therapy in the treatment of refractory MF lesions. METHODS: A total of 20 adult patients with early patch or plaque MF limited to less than 20% body surface area (BSA) involvement whose lesions were either stable or refractory to therapy for at least 8 weeks enrolled in an open-label pilot study. Tazarotene 0.1% gel was applied to MF lesions once daily for 24 weeks. Continued concomitant use of other medications such as low- to mid-potency topical corticosteroids was permitted for the alleviation of skin irritation. Global improvement, overall disease severity, percent BSA involvement, and pruritus were evaluated every 4 weeks. Up to 6 index lesions were followed up for area, plaque elevation, scaling, and erythema scores. Skin biopsy specimens were to be taken at baseline, week 8, and week 24. Evaluable specimens were stained with hematoxylin and eosin, CD8 antibody, and CD45RO antibody. RESULTS: In all, 20 patients enrolled, 19 received treatment, and 16 completed at least 4 weeks of topical treatment. By intent-to-treat analysis, 11 of 19 patients (58%) achieved at least a moderate (>50%) global improvement in BSA, and 35% of 99 index lesions cleared completely. Significant reductions (mean differences) were also found in the median lesional area score (-37, P =.0013), mean plaque elevation score (-.67, P =.016), mean scaling (-0.70, P =.033), and mean erythema score (-1.03, P =.002). Analysis of overall disease also disclosed statistical differences in percent of change for BSA involvement of 22% (P =.013) and of mean overall disease severity score of 34% (P =.011). Of 19 patients, 16 (84%) experienced mild or moderate local skin irritation manifested by peeling, erythema, burning, and tenderness that was managed successfully with topical steroids or reducing the frequency of treatment. Histopathology and immunohistochemistry results showed reductions in lymphocytic infiltrates and percentage of CD45RO(+) lymphocytes, and increases in the percentage of CD8(+) lymphocytes during the course of therapy. CONCLUSION: In this small pilot study, tazarotene 0.1% gel was a well-tolerated and effective adjuvant topical for the treatment of refractory MF lesions by clinical and histologic assessments.

Decreased immunogenicity of human fetal pancreas allografts following hyperbaric oxygen culture.

Human fetal pancreas (HFP) is a potential source of transplantable islets for the treatment of type 1 insulin-dependent diabetes mellitus (IDDM). Pretransplant culture techniques such as long-term culture, high-oxygen culture, UVB irradiation, and low-temperature culture have previously been used to reduce the immunogenicity of tissue for transplantation. In this study, we use hyperbaric oxygen culture (HOC) to modify MHC Class I expression on HFP and to reduce the immunological response of human peripheral blood mononuclear cells (PBMC) to HFP using a sponge matrix allograft model. To study the interaction of naïve PBMC with HOC-treated or untreated HFP allografts, sponges embedded with HFP tissue were implanted into the peritoneal cavity of NOD-SCID mice and injected with 1 x 10(7) freshly isolated human PBMC at the time of transplant. By day 14, human CD45 cells represented less than 2% of the cells recovered from the sponges implanted with HOC-treated HFP. In contrast, human CD45(+) cells represented nearly 15% (P =.0018) of the cells isolated from sponges implanted with conventionally cultured HFP grafts. Approximately 75% of the human CD45(+) cells from conventionally cultured HFP allografts were producing IFNgamma as determined by intracellular cytokine analysis. These data suggest that HOC treatment of HFP abrogates the activation and proliferation of PBMC. Pretransplant HOC treatment of islets is a simple technique that could be used to reduce immunogenicity and increase allograft survival while decreasing the requirement for immunosuppressive drugs.

Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise.

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% .VO(2 max). Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naïve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.

[Detection of myeloma cells in peripheral blood using multi-parameter flow cytometry: monitoring residual disease]. / Detekce myelomových bunek v periferní krvi vícerozmernou prutokovou cytometrií: monitorování zbytkové nemoci.

BACKGROUND: The aim of this study was to evaluate significance of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells counts in peripheral blood of patients with multiple myeloma for monitoring of the minimal residual disease. METHODS AND RESULTS: A triple-color flow cytometric analysis was used for this purpose. Peripheral blood of 29 patients with multiple myeloma who underwent high dose chemotherapy and autologous stem cells transplantation was repeatedly analysed. Counts of myeloma cells in peripheral blood were compared to serum monoclonal immunoglobulin concentration, serum calcium level, serum C-reactive protein, serum beta 2 microglobulin, and number of myeloma cells in bone marrow (morphology). From 29 patients in this study, 5 patients have relapsed. Patients in relapse had significantly higher counts of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells in peripheral blood than patients in remission (geometrical average: 12.41; 6.20; 14.45 cells/microliter versus 4.08; 2.87; 2.58 cells/microliter). The number of these cells correlated well with serum monoclonal immunoglobulin level and counts of myeloma cells in bone marrow. CONCLUSION: The authors conclude that the longitudinal multi-color flow cytometric analysis of CD 38(+2)45-54+, CD 38(+2)45-56+ and CD 38(+2)45-138+ cells in peripheral blood of patients with multiple myeloma is a useful method for evaluation of the disease activity. Significance of peripheral myeloma cells count for prediction of the relapse of the multiple myeloma remains to be evaluated.

Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.