Laser operation at 1.3microm of 2at.% doped crystalline Nd:YAG in a bounce geometry.

High efficiency and high power laser operation of highly-doped 2at.% crystalline Nd:YAG at 1.3microm is demonstrated in a diode-side pumped bounce amplifier configuration. A linearly polarized output power of 16.7W is obtained representing the highest power at 1.3microm achieved to date, to our knowledge, in a highly doped crystalline Nd:YAG laser. The system could deliver over 9W average output power in Q-switched operation with repetition rates from 20 - 160kHz. With quasi-continuous wave diode pumping 5.5mJ pulses at 100Hz repetition rate were achieved as well as 50ns pulses in Q-switched operation.

High efficiency laser operation of 2 at.% doped crystalline Nd:YAG in a bounce geometry.

High efficiency and high power operation of highly-doped 2 at.% crystalline Nd:YAG is demonstrated in a diode-side pumped bounce amplifier configuration. A linearly-polarized output power of 46.1W is obtained with 101W diode pumping representing the highest power achieved to date, to our knowledge, in a highly doped crystalline Nd:YAG laser. In a system operating at 19.1W output power, the slope efficiency was as high as 60%. With quasi-continuous wave diode pumping 11mJ pulses at 100Hz repetition rate were achieved and passive Q-switching with Cr(4+):YAG produced pulses with 12ns duration.

Microarray analysis of UVB-regulated genes in keratinocytes: downregulation of angiogenesis inhibitor thrombospondin-1.

BACKGROUND: Ultraviolet (UV) B light is an environmental mutagen that induces changes in cutaneous gene expression leading to immune suppression and carcinogenesis. Keratinocytes are a primary target for UVB. OBJECTIVE: To further delineate UVB-induced gene expression changes in keratinocytes. METHODS: cDNA microarray technology was utilized to examine gene expression in normal human KC (NHKC) following 20 mJcm(-2) UVB irradiation. Data was confirmed by semi-quantitative RT-PCR. RESULTS: Microarray analysis revealed 57 genes were upregulated, and 27 genes were downregulated, by at least two-fold following UVB. One downregulated gene was the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1). Semi-quantitative RT-PCR confirmed persistent downregulation of TSP-1 up to 18h following UVB. Microarray analysis also revealed upregulation of platelet-derived endothelial cell growth factor (PD-ECGF)--an angiogenesis activator. CONCLUSION: Our results suggest a gene expression mechanism by which UVB induces an angiogenic switch in keratinocytes. This may represent an important early event promoting neovascularization and growth of cutaneous neoplasms.

Modulation of the contact hypersensitivity response by AE-941 (Neovastat), a novel antiangiogenic agent.

BACKGROUND: AE-941 (Neovastat) is an angiogenesis inhibitor noted to have antiinflammatory properties. OBJECTIVE: We tested Neovastat in a contact hypersensitivity (CHS) model to determine the mechanism of action of its antiinflammatory effects. METHODS: Neovastat was orally administered (200 mg/kg/day) during the sensitization and challenge phases of a murine CHS assay and inflammatory responses were measured. Subsequent assays were performed on mice treated with Neovastat or Cortisone (120 mg/kg/day, IP) and differential mRNA expression of several pro- and antiinflammatory cytokines was quantified using RT-PCR. RESULTS: Neovastat decreased inflammation by 39% when administered during sensitization but did not alter the CHS response when given during the challenge phase. Neovastat significantly induced IL-10 expression in skin and skin-draining lymph nodes (49% and 45%, respectively) and decreased IFNgamma expression in the lymph nodes (35%). CONCLUSION: Antiinflammatory effects of Neovastat observed in CHS could be linked to modulation of cytokines early in the sensitization phase.

Malignant evolution of Schnitzler's syndrome--chronic urticaria and IgM monoclonal gammopathy: report of a new case and review of the literature.

Schnitzler's syndrome, initially described in 1974 is an uncommon condition defined by chronic urticaria and monoclonal IgM gammopathy. Additional features include fever of unknown origin, elevated ESR, bone pain and frequently a benign clinical course. We conducted a literature search of Medline, EMBASE and Cancerlit and found 56 cases of Schnitzler's syndrome reported to date. The absence of lymphoproliferative disease in this condition is typical, but nine patients have progressed to develop lymphoplasmacytic neoplasias, particularly Waldenstrom's macroglobulinemia (WM). Malignant evolution of Schnitzler's syndrome is a rare complication, but emphasizes the importance of long term follow-up and the need for these patients to undergo periodic assessment of the bone marrow and lymph nodes. Treatment of this condition is difficult, with varying response to corticosteroids and largely unsuccessful results with standard chemotherapy used for WM. We describe a case of Schnitzler's syndrome in a 50-year old man with lymphocytic aggregates in the bone marrow after 9 years of chronic urticaria, fever, arthralgias and bone pain. We review the clinical features and treatment, with emphasis on the hematologic aspects of this unusual condition.

Impaired ultraviolet-B-induced cytokine induction in xeroderma pigmentosum fibroblasts.

Xeroderma pigmentosum is a rare, autosomal recessive disease in which patients develop excessive solar damage at an early age and have a 1000-fold increased risk of developing cutaneous neoplasms. Xeroderma pigmentosum can be classified into seven complementation groups (A-G) with defects in different DNA nucleotide excision repair genes. Xeroderma pigmentosum patients also have impaired immune function including reduced natural killer cell activity and impaired induction of interferon-gamma. We hypothesized that altered cytokine induction may contribute to the immune defect in xeroderma pigmentosum patients. We examined cytokine mRNA expression after ultraviolet B irradiation using reverse transcriptase polymerase chain reaction in fibroblasts derived from five xeroderma pigmentosum patients in complementation groups A, C, and D and in complemented XP-A and XP-D cells. Cytokines interleukin-1beta and interleukin-6 displayed impaired ultraviolet B induction whereas interleukin-8 had normal induction in the xeroderma pigmentosum fibroblasts. Stable complementation of XP-A and XP-D cell lines increased ultraviolet-B-induced interleukin-1beta and interleukin-6 expression. These results demonstrate a deficient response of xeroderma pigmentosum fibroblasts to ultraviolet B in terms of cytokine interleukin-1beta and interleukin-6 induction but normal interleukin-8 induction and exhibit a role for DNA repair in cytokine induction.

Role of Th2 cytokines, RANTES and eotaxin in AIDS-associated eosinophilic folliculitis.

The pathogenesis of AIDS-associated eosinophilic folliculitis is still unknown. The expression of chemokines and Th2-type cytokines is increased in other conditions associated with tissue eosinophilia and in allergic reactions. We evaluated the mRNA expression by reverse transcriptase polymerase chain reaction of two Th2 cytokines (interleukin-4 and interleukin-5) and of two chemokines (RANTES and eotaxin) in the skin of 6 patients with AIDS-associated eosinophilic folliculitis; the tissue localization of eotaxin was shown by immunohistochemistry. We demonstrated the increased expression of interleukin-4, interleukin-5, RANTES and eotaxin in lesional skin of the patients compared to normal skin of HIV+ individuals. We concluded that a Th2 pattern is present in AIDS-associated eosinophilic folliculitis. The cytokine milieu in this disease may favour a Th2 immune response to an unknown antigen, whereby RANTES and eotaxin act in synergy with interleukin-4 and interleukin-5 to mediate tissue inflammation.

Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies.

Contact hypersensitivity (CHS), a dendritic-cell (DC)-dependent, T-cell-mediated skin immune response to reactive haptens, has been a subject of intense research for many years. The molecular mechanisms underlying CHS are complicated and are not fully understood. During the past few years, varieties of gene-targeted knockout mice have been used in the study of CHS. Such studies have contributed significantly to our understanding of the mechanisms responsible for the initiation of CHS. This review focuses on insights into molecular requirements for CHS gained from knockout studies.

Rethinking the role of tumour necrosis factor-alpha in ultraviolet (UV) B-induced immunosuppression: altered immune response in UV-irradiated TNFR1R2 gene-targeted mutant mice.

BACKGROUND: Ultraviolet (UV) B-induced immunosuppression, implicated in the pathogenesis of skin cancers, is postulated to be mediated in part by cis-urocanic acid (cis-UCA) via tumour necrosis factor (TNF)-alpha. TNF-alpha produces morphological changes in Langerhans cells indistinguishable from those induced by UVB exposure and antibodies against TNF-alpha have been demonstrated to inhibit UVB-induced immunosuppression in vivo. OBJECTIVES: To clarify further the role of TNF-alpha in UVB-induced immunosuppression and in cis-UCA immunosuppression. METHODS: We performed a contact hypersensitivity (CHS) assay on gene-targeted mutant mice (TNFR1R2-/-) lacking genes for both receptors (p55 and p75) for TNF-alpha. Mice were either irradiated with UVB or injected intradermally with cis-UCA, sensitized with 2,4-dinitrofluorobenzene, challenged on the ears and the response was measured. RESULTS: The TNFR1R2-/- mice showed hyporesponsiveness in the CHS response compared with wild-type (P < 0.001), confirming the proinflammatory role of TNF-alpha. However, significant suppression of CHS was seen after irradiation and after cis-UCA injection in both locally (sensitization on irradiated site; P < 0.05) and systemically (sensitization on non-irradiated site; P < 0.05) sensitized wild-type and gene-targeted mice. CONCLUSIONS: These results demonstrate that TNF-alpha signalling is only partially involved in UVB-induced immunosuppression and does not play a major part in the cis-UCA immunosuppression mechanism.

cis-Urocanic acid does not induce the expression of immunosuppressive cytokines in murine keratinocytes.

trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10-100 micrograms/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, beta-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-alpha mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 micrograms/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-alpha and TGF-beta, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity.

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.

The use of plasmapheresis and immunosuppression in the treatment of pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris is an autoimmune blistering disease for which the mainstay of treatment is systemic corticosteroids and immunosuppressants. This therapy had reduced the mortality of pemphigus; however, it is associated with significant morbidity. OBJECTIVE: The objective of this study was to assess the group's experience with plasmapheresis in the treatment of pemphigus vulgaris and report on its utility. METHODS: Seven patients with severe or resistant pemphigus vulgaris underwent a series of 5 plasma exchanges over an average of 8 days. Immunosuppressive drugs were administered immediately after plasmapheresis to prevent the "rebound" flare of disease that can occur after plasmapheresis. RESULTS: Remission was induced in 4 patients, partial remission was induced in 2 patients, and 1 patient continues to have active disease. CONCLUSION: This study suggests that plasmapheresis is a useful intervention in patients with pemphigus vulgaris who are not responding to standard therapy or who require unacceptably high doses of steroids or immunosuppressants.

The effect of VAS972 on allergic contact hypersensitivity.

BACKGROUND: Contact hypersensitivity (CHS) is a Th1-mediated immune response that can be down-regulated by immunosuppressive agents such as cyclosporine and environmental stimuli such as ultraviolet light. Recently, an immunomodulation therapy, VAS972, has been developed which is believed to down-regulate the Th1 arm of the immune response. This VAS972 involves modifying autologous blood by controlled exposure to the oxidizing agent ozone and UVC light, at an elevated temperature ex vivo. The processed blood is then administered by intramuscular injection. OBJECTIVE: To further evaluate the immune modulating effect of VAS972. METHODS: We examined the effect of VAS972 treatment on CHS. Contact hypersensitivity was induced with dinitrofluorobenzene (DNFB) in animals receiving VAS972- processed blood, control blood, or saline. A preliminary study was also conducted to evaluate the effect of plasma and cellular fractions of processed blood. RESULTS: Mice injected with VAS972-processed blood demonstrated a significantly lower (46%) CHS response than controls. Histologic examination of challenged ear skin from control mice displayed edema with a significant lymphocytic infiltration, whereas animals administered processed blood demonstrated a reduction in lymphocytic infiltration. Mice injected with either plasma or the cellular fraction of the VAS972-treated blood also demonstrated a significant suppression (49% and 41%, respectively). CONCLUSION: The results of this study demonstrated that VAS972 suppresses CHS and cellular infiltration. Furthermore, the plasma and cellular components of the VAS972 treatment were also able to induce immunosuppression. This further supports the hypothesis that VAS972 down-regulates the Th1 arm of the immune response.

Immunomodulatory and pharmacologic properties of imiquimod.

Imiquimod, an imidazoquinoline amine, is an immune response modifier recently approved for the topical treatment of external genital and perianal warts. Although the majority of immunomodulatory agents available or in development inhibit pathways involved in immune activation, imiquimod is unique in that it activates immune function. The exact mechanism of imiquimod's antiviral activity is unknown; however, its effects are likely to be related to its immunomodulating properties. Although in vitro studies have shown that imiquimod has no direct antiviral effects, the drug does exhibit antiviral and antitumor effects in vivo through induction of cytokines and enhancement of cell-mediated cytolytic antiviral activity. Imiquimod stimulates the innate immune response through induction of cytokines, and the cellular arm of acquired immunity through induction of interferon-alpha (IFN-alpha), IFN-gamma, and interleukin-12. Results from animal studies have indicated a possible use for imiquimod in the prevention and treatment of herpes simplex virus infection. In addition, recent studies demonstrated that imiquimod activates Langerhans' cells and enhances allergic contact hypersensitivity.

Molecular mechanism of ultraviolet-induced keratinocyte apoptosis.

This article reviews advances in the study of the molecular mechanisms for ultraviolet (UV)-induced keratinocyte apoptosis, with particular reference to the cytokines tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL). TNF-alpha and FasL induce their respective receptors and then activate caspase enzymes that are critically involved in the apoptotic process. This activation is further amplified by intracellular mitochondria-associated mechanisms. Using gene-targeted knockout mice lacking either the TNF-Rp55 or the TNF-Rp75, we have shown that TNF-alpha plays an important role in UV-induced keratinocyte apoptosis via TNF-Rp55. TNF-Rp55 shares homology with Fas and contains an intracellular death domain. UV seems to directly stimulate cross-linking of Fas, resulting in the engagement of the death machinery. Fas-associated death domain protein (FADD) acts as an adapter protein in both the TNF-Rp55 and Fas death-inducing cascades and is responsible for downstream signal transduction by recruiting caspases. Moreover, signaling of p53 contributes to the induction of apoptosis by regulating Bcl-2 family expression and increasing surface Fas expression. In addition to induction mechanisms of apoptosis, there are numerous inhibitory molecules that play a role in restricting the apoptotic pathway. Thus, the ultimate determination of whether or not a cell undergoes apoptosis after UV radiation is based on the balance between agonist and antagonist pathways.

Screening for azathioprine toxicity: a pharmacoeconomic analysis based on a target case.

The risk of azathioprine-induced myelosuppression can be predicted by detecting patients with intermediate or low thiopurine methyltransferase (TPMT) activity. Population studies have shown that 89% of whites have high TPMT activity, 11% have intermediate TPMT activity, and 0.3% have low TPMT activity. Three specific mutations in the TPMT gene that cause decreased TPMT activity have recently been identified. Patients homozygous for the TPMT mutations have low TPMT activity, and patients heterozygous for TPMT mutations have intermediate TPMT activity. This has led to the development of a technique for TPMT genotype analysis that will identify patients at risk of azathioprine-induced myelosuppression. We report a case of a patient with bullous pemphigoid who experienced azathioprine-induced myelosuppression and who was later found to be homozygous for TPMT mutant alleles. Using the cost of treatment required for this patient and the estimated population prevalence of TPMT mutations, we examined the cost impact of screening for TPMT mutations in all patients being considered for azathioprine therapy. We calculated that screening is cost-neutral assuming patients homozygous for TPMT mutations experience myelosuppression, and that it is cost-beneficial assuming patients heterozygous for TPMT mutations also experience myelosuppression while receiving azathioprine. Screening patients for TPMT mutations will reduce the risk of azathioprine-induced myelosuppression, and our study suggests that it may also be a cost-attractive strategy.

Open-label study to evaluate the healing rate and safety of the Profore Extra Four-Layer Bandage System in patients with venous leg ulceration.

BACKGROUND: Venous ulcers are increasing in prevalence, especially since these are observed more frequently in the elderly, and the number of individuals in this age group is becoming a larger portion of the population. OBJECTIVE: To determine the healing rate and safety of the Profore Extra Four-Layer Bandage System in the management of venous leg ulcers. METHODS: In an open-label study, patients aged 18 years or older with venous leg ulcers were treated with a high compression four-layer bandage system in which a hydrocellular dressing was placed in contact with the wound. The combination is designated the "Profore Extra Four-Layer Bandage System." Follow-up visits took place weekly unless there was heavy exudation from the ulcer or if there was marked edema of the leg at the start of the study requiring reapplication of the bandage system. RESULTS: Fifteen patients were entered into the study (men 8, women 7, mean age 66 years, mean duration of ulcers 1.3 years). Thirteen of the 15 patients completed the study, with two withdrawals. In one patient who withdrew, the ulcer became infected and required treatment with antibiotics. The other termination from the study occurred for reasons unrelated to treatment. The ulcer in this patient healed in 7 weeks. Ten of the 13 patients (77%) who completed the study, and 10 (67%) of 15, who had enrolled experienced complete (100%) healing. Healing of > 80% of the ulcers occurred in 11 of 13 patients (85%) who completed the study and in 12 (80%) of 15 enrolled patients. No patient experienced a study-related adverse event. One patient developed contact dermatitis and was later found to have stasis dermatitis. It is unclear whether the initial event was contact or stasis dermatitis. CONCLUSION: In this open-label study, a high compression system, using the Profore Extra Four-Layer Bandage with a hydrocellular dressing in contact with the wound, was found to be effective and safe for the treatment of venous leg ulcers.

Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essential for fibronectin binding.

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1(21-34) and D3(20-33), which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3(20-33) immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.