PD-1 AND TIM-3 BLOCKING CANNOT ENHANCE APOPTOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS INDUCED BY PERIPHERAL BLOOD CD8+ T CELLS.

AIM: Given the invaluable success of immune checkpoint inhibitors for tumor immunotherapy, in this study, the effect of programmed cell death 1 (PD-1) and T cell immunoglobulin-3 (TIM-3) blocking was investigated to induce apoptosis of leukemic cells by exhausted CD8+ T cells in patients with chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: Peripheral blood CD8+ T cells were positively isolated from 16 CLL patients using magnetic beads separation method. Isolated CD8+ T cells were treated with either blocking anti-PD-1, anti-TIM-3 and isotype-matched control antibodies and then co-cultured with CLL leukemic cells as target. The percentage of apoptotic leukemic cells and the expression of apoptosis-related genes were evaluated by flow cytometry and real-time polymerase chain reaction methods, respectively. Interferon gamma and tumor necrosis factor alpha concentration was also measured by ELISA. RESULTS: Flow cytometric analysis of apoptotic leukemic cells indicated that the blockade of PD-1 and TIM-3 did not significantly enhance the apoptosis of CLL cells by CD8+ T cells, which then were confirmed by analysis of BAX, BCL2 and CASP3 gene expression, which was similar in blocked and control groups. No significant difference was found between blocked and control groups in terms of interferon gamma and tumor necrosis factor alpha production by CD8+ T cells. CONCLUSION: We concluded that the blockade of PD-1 and TIM-3 is not an effective strategy to restore the function of CD8+ T cells in CLL patients at the early clinical stages of the disease. Further in vitro and in vivo studies are needed to more address the application of immune checkpoint blockade in CLL patients.

Corneal biomechanical properties after soft contact lens wear measured on a dynamic Scheimpflug analyzer: A before and after study.

OBJECTIVES: To evaluate the corneal biomechanics before and after daily use of contact lenses (CLs), measured by Scheimpflug-based devices. METHODS: This prospective clinical study includes participants who were scheduled to use CLs daily for refractive error. The biomechanical parameters were measured by the Corneal Visualization Scheimpflug Technology (Corvis ST) before and one month after using the soft CLs. RESULTS: Twenty-three subjects (46 eyes), including 16 female (76.2%) with a mean age of 28±7.29 years, were enrolled. There was no significant difference among biomechanical factors measured before and after contact lens wear (P>0.05). Using regression analysis of the biomechanical markers, we found a statistically significant association between second applanation length (A2 length) (P=0.001), highest concavity radius (HCR) (P=0.05), deflection amplitude ratio (DA_ratio) (P=0.05) and integrated radius (P<0.001) with age. Regarding spherical equivalent, we found a statistically significant association between central corneal thickness (CCT) (P=0.05), A2 length (P=0.03) and stiffness parameter at first applanation (SPA1) (P=0.02). CONCLUSIONS: We did not find a significant difference in terms of corneal biomechanical parameters between baseline and month 1; but regression analyses showed a statistically significant association between A2 length, HCR, DA_ratio, integrated radius, CCT and SPA1 and certain subject characteristics.

Investigation of hyperelastic models for nonlinear elastic behavior of demineralized and deproteinized bovine cortical femur bone.

In this study, the hyperelastic models of demineralized and deproteinized bovine cortical femur bone were investigated and appropriate models were developed. Using uniaxial compression test data, the strain energy versus stretch was calculated and the appropriate hyperelastic strain energy functions were fitted on data in order to calculate the material parameters. To obtain the mechanical behavior in other loading conditions, the hyperelastic strain energy equations were investigated for pure shear and equi-biaxial tension loadings. The results showed the Mooney-Rivlin and Ogden models cannot predict the mechanical response of demineralized and deproteinized bovine cortical femur bone accurately, while the general exponential-exponential and general exponential-power law models have a good agreement with the experimental results. To investigate the sensitivity of the hyperelastic models, a variation of 10% in material parameters was performed and the results indicated an acceptable stability for the general exponential-exponential and general exponential-power law models. Finally, the uniaxial tension and compression of cortical femur bone were studied using the finite element method in VUMAT user subroutine of ABAQUS software and the computed stress-stretch curves were shown a good agreement with the experimental data.

Effect of lemon verbena powder and vitamin C on performance and immunity of heat-stressed broilers.

An experiment was conducted to investigate the effect of lemon verbena powder and vitamin C on performance and immunity of broilers under heat stress. The experiment was carried out with a total of 160-day-old male Ross 308 broiler chickens in a completely randomized design. From 25 days of age, the birds were assigned to four diets including control diet and treatment diets which were supplemented with 2 levels of lemon verbena (0.5% and 1.0%) and one level of vitamin C (250 mg/kg) in 16-floor pens with 10 chickens each and reared to 42 days of age. To induce chronic heat stress, birds were exposed to an ambient temperature of 35 ± 2 °C for 8 h daily (from 09:00 AM until 17:00 PM) between 25 and 42 days of age. At the end of experiment, one chick/pen was randomly selected, and the performance and blood parameters were evaluated. Dietary supplementation of 1.0% lemon verbena increased (p < 0.05) average weight gain and feed intake by 5.81% and 3.29%, and reduced feed conversion ratio by 2.59% respectively compared to control group. Birds fed diets containing 1.0% lemon verbena had significantly higher relative weight of bursa of fabricius and breast (p < 0.05). LDL decreased by 15.85% and 17.57%, for birds fed 0.5% and 1.0% lemon verbena respectively. The ratio of heterophyl to lymphocyte was reduced (p < 0.05) by 20.68% via significant decrease in heterophyl by 15.55% and significant increase in lymphocyte by 4.51% in birds fed lemon verbena at the rate of 1.0% compared to those fed the control diet. 1.0% lemon verbena and vitamin C elevated (p = 0.0005) the level of glutathione peroxidase by 51.81% and 27.90%, respectively. In conclusion, lemon verbena and vitamin C improved some performance data and blood metabolites which somehow suppressed the negative effects of heat stress.

Synergistic effect between prelimbic 5-HT3 and CB1 receptors on memory consolidation deficit in adult male Sprague-Dawley rats: An isobologram analysis.

The serotonergic system has often been defined as a neuromodulator system, and is specifically involved in learning and memory via its various receptors. Serotonin is involved in many of the same processes affected by cannabinoids. The present study investigated the influence of bilateral post-training intra-prelimbic (PL) administrations of serotonergic 5-hydroxytryptamine type-3 (5-HT3) receptor agents on arachidonylcyclopropylamide (ACPA) (cannabinoid CB1 receptor agonist)-induced amnesia, using the step-through inhibitory avoidance (IA) task to assess memory in adult male Sprague-Dawley rats. The results indicated that sole intra-PL microinjection of ACPA (0.1 and 0.5 µg/rat) and 5-HT3 serotonin receptor agonist (m-Chlorophenylbiguanide hydrochloride, m-CPBG; 0.001, 0.01 and 0.1 µg/rat) impaired, whereas Y-25130 (a selective 5-HT3 serotonin receptor antagonist; 0.001 and 0.01 and 0.1 µg/rat) did not alter IA memory consolidation, by itself. Moreover, intra-PL administration of subthreshold dose of m-CPBG (0.0005 µg/rat) potentiated, while Y-25130 (0. 1 µg/rat) restored ACPA-induced memory consolidation deficit. The isobologram analysis showed that there is a synergistic effect between ACPA and m-CPBG on memory consolidation deficit. These findings suggest that 5-HT3 receptor mechanism(s), at least partly, play(s) a role in modulating the effect of ACPA on memory consolidation in the PL area.

Clinical outcomes of MyoRing implantation in keratoconic eyes by using the Femtosecond laser technology.

Purpose: To assess the outcomes of the insertion of the MyoRing (DIOPTEX, GMBH, Linz, Austria) by applying the Femtosecond Laser Technology (FLT) method in eyes that have keratoconus problem. Methods: A prospective, nonrandomized, clinical examination was managed. 27 eyes of 15 subjects with stable keratoconus (6 females and 9 males), with lifetimes differing from 14 to 49 years were involved. All subjects have problems about decreased fine-accurate ocular awareness, lens intolerantness or trouble, and also the middle corneal height larger than 350 micrometers. MyoRing additions of about 320 micrometers into height and 2.5 mm into radius were inserted in each subject inside an Intrastromal Corneal Pocket using(ICP) formed with applying FLT. Ocular, refractive errors, corneal shape, and pachymetry changes were assessed throughout a 6-months follow-up period. Results: The average UDVA (uncorrected distance visual acuity) notably increased originating at 1.73 ± 0.53 LogMAR preoperationally to 0.54 ± 0.40 LogMAR post-operationally. The average CDVA (corrected distance ocular awareness) notably increased from 0.59 ± 0.47 LogMAR preoperationally to 0.27 ± 0.16 LogMAR post-operationally. The change in the average UDVA and CDVA was analytically meaningful (P< 0.000). The average reduction in the average keratometry from preoperational to 26 weeks post operational was -6.41 ± 3.62 D. This change was analytically meaningful (P< 0.000). The average lowest and highest keratometry amounts were similarly analytically notable at shorter than 26 weeks preoperatively. A notable increase in UDVA and CDVA was observed 26 weeks following the operation, that was compatible with the notable decrease in sphere and cylinder. Moreover, a notable corneal straightening with an average amount of -6.41 ± 3.62 diopters (D) was determined. Conclusion: MyoRing implantation employing FLT would be a harmless, effective, and predictable method to treat selected subjects of keratoconus, being a helpful choice for the therapy of keratoconus.

Toll-like receptor 7 stimulation promotes autoimmune diabetes in the NOD mouse.

AIMS/HYPOTHESIS: The role of Toll-like receptor 7 (TLR7), a sensor of viral and self RNA, in promoting autoimmune diabetes remains unclear. Our goal was to determine the effect of TLR7 stimulation on the priming and activation of diabetogenic CD8(+) T cells. METHODS: We explored the effects of CL097 (TLR7/8 agonist) and immunoregulatory sequence 661 (IRS661, TLR7 inhibitor) on bone marrow-derived dendritic cells (BMDCs), diabetogenic CD8(+) T cell function and autoimmune diabetes onset in NOD and 8.3 NOD T cell receptor transgenic mice (8.3 NOD mice). RESULTS: TLR7 stimulation of NOD BMDCs increased activation and production of proinflammatory cytokines. In vivo administration of CL097 activated T cells and dendritic cells and increased levels of proinflammatory cytokines and type 1/2 IFNs in NOD mice. In vivo antigen-specific cytotoxicity studies revealed enhanced cytotoxicity against islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, an islet autoantigen) peptide pulsed targets in NOD mice treated with CL097 plus CD40 agonist. This combination treatment accelerated the onset of autoimmune diabetes in 8.3 NOD mice. Likewise, topical treatment of NOD mice with a TLR7 agonist accelerated diabetes onset. Spontaneous disease in 8.3 NOD mice and accelerated disease in CL097+CD40 agonist-treated 8.3 NOD mice were delayed by IRS661 treatment, which is associated with inhibition of the endogenous upregulation of IFN-α levels within the pancreatic lymph nodes. CONCLUSIONS/INTERPRETATION: TLR7 stimulation accelerates the spontaneous onset of autoimmune diabetes in 8.3 NOD and NOD mice. Conversely, TLR7 inhibition prevents the early events associated with diabetogenesis.

Cutaneous lupus erythematosus: recent lessons from animal models.

Cutaneous lupus erythematosus (CLE) may present as a clinically heterogeneous group of lupus-specific skin lesions that have common histopathological findings. Determination of the immunopathological sequence of events in this group of disorders has been challenging for dermatologists and immunologists but is vital for therapeutic targeting. We review animal models in which different aspects of immune alteration in CLE have been addressed. The MRL/lpr mouse develops spontaneous skin disease with some features of CLE. Study of this strain and related gene-manipulated strains has revealed roles for multiple cytokines, including interleukin (IL)-6, IL-18, and IL-21, in disease pathogenesis. A role for the growth factor colony stimulating factor 1 and the inflammatory protein high-mobility group box 1 has also been suggested. We discuss potential novel treatment options suggested by these models.

Type 1 interferon signature in the scalp lesions of alopecia areata.

BACKGROUND: Autoimmune attack of the bulbar region of anagen phase hair follicles by CD8+ T cells and Th1 cytokines has been proposed to result in hair loss in alopecia areata (AA). The initiating stimuli are unknown. As interferon-alpha therapy may trigger AA, we propose that type 1 interferons are involved in the induction of disease. OBJECTIVES: To compare lesional scalp from patients with AA with scalp lesions of cutaneous diseases associated with local type 1 interferon-related protein expression. METHODS: Lesional scalp of patients with AA, discoid lupus erythematosus, lichen planopilaris and androgenetic alopecia was examined by immunohistochemistry for expression of the type 1 interferon-inducible myxovirus protein A (MxA), the chemokine receptor CXCR3, and the cytotoxic proteins granzyme B (GrB) and T-cell intracytoplasmic antigen 1 (TiA-1). RESULTS: MxA was expressed in the intradermal and subcutaneous compartments of the hair follicle including sebaceous glands in inflammatory AA similar to lesions of cicatricial alopecia (discoid lupus erythematosus, lichen planopilaris) but not in the epidermal compartment of AA, and not at all in noninflammatory AA or androgenetic alopecia. The location of CXCR3-expressing cells correlated with MxA expression. The inflammatory cells around the hair follicle in AA included a lower number of GrB+ and TiA-1+ cells compared with cicatricial alopecia and demonstrated predominant TiA-1+ expression. CONCLUSIONS: We demonstrate the expression of type 1 interferon-related proteins in the inflammatory lesions of AA. The distribution pattern of the interferon signature and cytotoxicity-associated proteins in AA differs from cicatricial alopecia.

The treatment of atopic dermatitis with licorice gel.

Glycyrrhiza glabra L. has been used in herbal medicine for skin eruptions, including dermatitis, eczema, pruritus and cysts. The effect of licorice extract as topical preparation was evaluated on atopic dermatitis. The plant was collected and extracted by percolation with suitable solvent. The extract was standardized, based on Glycyrrhizinic acid by using a titrimetry method. Different topical gels were formulated by using different co-solvents. After standardizing of topical preparations, the best formulations (1% and 2%) were studied in a double-blind clinical trial in comparison with base gel on atopic dermatitis over two weeks (30 patients in each group). Propylene glycol was the best co-solvent for the extract and Carbopol 940 as gelling agent showed the best results in final formulations. The quantity of glycyrrhizinic acid was determined 20.3% in the extract and 19.6% in the topical preparation. Two percent licorice topical gel was more effective than 1% in reducing the scores for erythema, oedema and itching over two weeks (p<0.05). The results showed that licorice extract could be considered as an effective agent for treatment of atopic dermatitis.

Expression of 27 KD, 65 KD and 72/73 KD heat shock protein in atopic dermatitis: comparison with those in normal skin and contact dermatitis.

The expression of Heat Shock Protein (HPS) 72/73, HSP65 and HSP27 in skin lesions of atopic dermatitis (n = 21) was studied and compared with that in contact dermatitis (n = 18) and normal skin (n = 9). Keratinocytes in the whole epidermis expressed both HSP65 and HSP72/73 with a membranous, cytoplasmic or nuclear/perinuclear staining pattern much more intensely in atopic dermatitis than in contact dermatitis and normal subjects. In approximately half of the subjects with atopic dermatitis, infiltrating cells in the dermis expressed HSP65 and HSP72/73; this was not observed in contact dermatitis. HSP27 was expressed in the upper epidermis with a cytoplasmic or nuclear/perinuclear staining pattern in all groups. HSP27 was not expressed by infiltrating cells. A clinical evaluation of atopic dermatitis showed that more severe types of atopic dermatitis expressed more intense expression of HSP65 and HSP72/73, but not HSP27, in their skin lesions. These findings suggested that HSP65 and HSP72/73 may play roles in the pathogenesis of atopic dermatitis.

Signal transducer and activator of transcription 6 is essential in the induction of contact hypersensitivity.

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6(-/)-) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6(-/)- mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6(-/)- mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate-modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6(-/)- mice, whereas the expression of interferon gamma was the same in STAT6(-/)- and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6(-/)- mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.

Heat shock proteins in the pathogenesis of inflammatory skin diseases.

Heat shock proteins (HSPs) are highly conserved molecules and distributed widely in nature. They are also distributed in the skin, however, only limited information is available on the role of HSPs in the skin diseases. Immunohistochemical study of HSPs in the skin revealed that HSPs are differently expressed in the epidermal cells of patients with systemic lupus erythematosus (SLE), atopic dermatitis, graft versus host disease (GVHD) and so on. In normal healthy skin HSPs are constantly expressed in the epidermal cells. HSPs are expressed in the skin according to the influence of both external and internal milieu of the diseased conditions. Cytokines released in the skin strongly affect to express HSPs in epidermal cells. HSPs expressed in the skin can be targets for infiltrated T cells to modulate immune response of skin diseases. Roles of HSPs in the pathogenesis of SLE, GVHD, atopic dermatitis and psoriasis are discussed in this review. HSPs play an important role in the pathogenesis of many inflammatory skin diseases. They can be the molecules to evaluate both diseased conditions and inflammatory process of the skin diseases.

IL-4 inhibits the migration of human Langerhans cells through the downregulation of TNF receptor II expression.

The migration of Langerhans cells is an initial event in the sensitization phase of contact sensitivity. Langerhans cells travel from the epidermis to the regional lymph node, and can be variously modulated in the skin where many cytokines are released from epidermal cells, dermal cells, T helper (Th) cells, and other inflammatory cells during the sensitization and elicitation phase of contact dermatitis, and thus induce an altered inflammatory skin reaction. The modulatory effect of the cytokines released in the skin, such as IL-1beta, GM-CSF, and TNF-alpha as epidermal cytokines, IL-2, IL-12, and IFN-gamma as Th1 type cytokines, and IL-4 and IL-10 as Th2 type cytokines, was analyzed using the chemotactic chamber method in this study. Both GM-CSF and TNF-alpha induced the migration of human Langerhans cells in vitro, whereas IL-1beta, IL-2, IL-10, IL-12, and IFN-gamma had no effect on Langerhans cell migration. In contrast, IL-4 inhibited Langerhans cell migration in a dose dependent manner. The inhibitory activity of IL-4 was reversed by both anti-human IL-4 monoclonal antibody and anti-human IL-4 receptor monoclonal antibody. IL-4 inhibited the Langerhans cell migration induced by both TNF-alpha and GM-CSF. Furthermore, anti-TNF-RII monoclonal antibody inhibited both random migration and the migration induced by TNF-alpha, but not that induced by GM-CSF. A reverse-transcriptase-polymerase chain reaction and fluorescence-activated cell sorter analysis revealed that TNF-alpha up-regulated and IL-4 downregulated the TNF receptor II (TNF-RII) expression of Langerhans cells at both the mRNA and the protein levels. The pretreatment of Langerhans cells with TNF-alpha enhanced the migration of Langerhans cells and the expression of TNF-RII. After pretreating Langerhans cells with TNF-alpha, IL-4 inhibited both the migration of Langerhans cells and the expression of TNF-RII in a time dependent manner. These results indicate that IL-4 inhibits the migratory activity of Langerhans cells by downregulating the expression of TNF-RII in human Langerhans cells and thereby modulates the immune response in the skin.

A case of Post-kala-azar dermal leishmaniasis.

Only a few cases of Post-kala-azar dermal leishmaniasis have been reported in Japan, especially recently. We describe a case of a 32-year-old woman who developed rose-colored nodules on her forearms two years after Kala-azar. A skin biopsy specimen from a nodule revealed not only granulomatous changes but also many amastigotes of Leishmania donovani in macrophages. Rose-colored nodules were also distributed on her face and neck. Treatment with antimony compound was very effective.

Analysis of 70 KD heat shock protein (HSP70) expression in the lesional skin of lupus erythematosus (LE) and LE related diseases.

Biopsied specimens from skin lesions of SLE were studied for expression of 70 KD heat shock protein (HSP70). The pattern of HSP70 expression in SLE was diffuse in whole epidermis, hair follicles, and sweat gland cells and rather more intense than that in other control groups or normal skin. No significant differences in HSP70 expression were observed between sun-exposed and protected areas of SLE skin lesions. Unlike SLE, reduced or no expression of HSP70 was observed in skin lesions of DLE. In tissue culture, UVB radiation in vitro induced relatively intense expression of HSP70 in the nuclear area of keratinocytes. A few gamma delta T cell receptor positive cells which might respond to HSP70 expressing cells were detected in the basal layer of skin lesions of diseases. These studies suggest that aberrant expression of HSP70 in skin lesions of SLE might contribute to both skin lesions and antibody formation in SLE.