The pathogenic activity of anti-desmoglein autoantibodies parallels disease severity in rituximab-treated patients with pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune blistering disease mediated by IgG autoantibodies targeting desmogleins (Dsgs). The anti-CD20 monoclonal antibody rituximab is increasingly used in corticosteroid-resistant PV patients. In a subset of rituximab-treated patients in remission, high ELISA index values have been reported; however, their significance remains so far unclear. OBJECTIVE: To address the discrepancy between anti-Dsg3 serum antibody titers and disease severity. MATERIALS & METHODS: 6 rituximab-treated PV patients were prospectively followed-up for two years and anti-Dsg3 autoantibodies levels and pathogenic activity were measured. RESULTS: All patients achieved complete remission without any serious side effects. Both anti-Dsg3 autoantibodies (p = 0.031) and their pathogenic activity (p = 0.003) were significantly related to disease severity. However, in selected patients, the dissociation index was a more sensitive indicator for PV clinical activity than the ELISA index. CONCLUSION: Our findings have demonstrated the existence of non-pathogenic autoantibodies in PV patients in remission, establishing the basis for the design of a system able to precisely monitor the course of disease.

Betapapillomavirus in multiple non-melanoma skin cancers of Netherton syndrome: Case report and published work review.

Netherton syndrome (NS) is a rare genetic disease presenting with ichthyosiform erythroderma, hair alterations and atopy. NS is due to SPINK5 gene mutations, which cause absent or decreased expression of the encoded protein lymphoepithelial Kazal-type-related inhibitor (LEKTI) in all stratified epithelia. We report a 43-year-old man affected with NS, who developed several squamous and basal cell carcinomas on the face, ears and scalp and papillomatous lesions of hips, groin and genitoanal area. Molecular analysis of the SPINK5 gene revealed homozygosity for the recurrent mutation c.238dupG. Human papillomavirus (HPV) DNA detection and genotyping on patient skin carcinomas and hyperplastic lesions found betapapillomavirus DNA in 10 of 12 (83%) carcinomas and in a hip papilloma, with multiple betapapillomavirus types being identified. Immunohistochemistry showed upregulated expression of p16(INK4a) protein in nine of 12 (75%) patient carcinomas, in line with findings reported in HPV-related cancers. LEKTI and filaggrin immunostaining was strongly decreased in patient skin. A published work search for NS cases with skin cancers and HPV infection identified 15 NS patients, five of them showing mucosal or cutaneous HPV infection. Overall, our results confirm the increased susceptibility to skin carcinomas of some NS patients and provide further evidence of an association between HPV and non-melanoma skin cancers in NS. The highly impaired skin barrier function, hallmark of NS, could facilitate HPV infection, in turn increasing the risk for cancer development.

Upregulation of the inhibitory receptor ILT4 in monocytes from septic patients.

Sepsis-induced immune dysfunction is a complex phenomenon that involves both innate and adaptive responses. Upregulation of the inhibitor receptor named immunoglobulin like transcript 4 (ILT4) is crucial to the tolerogenic function of monocytes. Here, ILT4 expression, endotoxin-induced IL-12 and IL-10 production and CD86 expression were investigated in circulating monocytes from 16 patients with severe sepsis and 16 age and sex matched controls. We found that monocytes from patients with severe sepsis express significantly higher levels of ILT4 than monocytes from controls. Upregulation of ILT4 expression appeared to be induced by soluble factors present in the serum of septic patients and directly correlated with the degree of organ dysfunction. ILT4(+) monocytes from septic patients also displayed an alteration in the cytokine response to endotoxin stimulation characterized by reduced IL-12 production and increased IL-10 production, and a reduced expression of the costimulatory molecule CD86. In conclusion, the increased ILT4 expression and IL-10 production and the decreased CD86 expression and IL-12 production indicate that during sepsis monocytes undergo substantial modulation of the surface and cytokine phenotype. These phenotypic changes may interfere with the antigen presenting cell activity of monocytes, which may contribute to the impairment of adaptive immune responses that takes place during sepsis.

Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface.

Pemphigus vulgaris (PV) is an autoimmune blistering disease of skin and mucous membranes caused by autoantibodies to the desmoglein (DSG) family proteins DSG3 and DSG1, leading to loss of keratinocyte cell adhesion. To learn more about pathogenic PV autoantibodies, we isolated 15 IgG antibodies specific for DSG3 from 2 PV patients. Three antibodies disrupted keratinocyte monolayers in vitro, and 2 were pathogenic in a passive transfer model in neonatal mice. The epitopes recognized by the pathogenic antibodies were mapped to the DSG3 extracellular 1 (EC1) and EC2 subdomains, regions involved in cis-adhesive interactions. Using a site-specific serological assay, we found that the cis-adhesive interface on EC1 recognized by the pathogenic antibody PVA224 is the primary target of the autoantibodies present in the serum of PV patients. The autoantibodies isolated used different heavy- and light-chain variable region genes and carried high levels of somatic mutations in complementary-determining regions, consistent with antigenic selection. Remarkably, binding to DSG3 was lost when somatic mutations were reverted to the germline sequence. These findings identify the cis-adhesive interface of DSG3 as the immunodominant region targeted by pathogenic antibodies in PV and indicate that autoreactivity relies on somatic mutations generated in the response to an antigen unrelated to DSG3.

Vitamin D3 modulates T lymphocyte responses in hepatitis C virus-infected liver transplant recipients.

BACKGROUND: Aim of the present study was to investigate whether 1,25(OH)(2)D(3) (Vitamin D3) modulates T lymphocyte functions in patients transplanted for hepatitis C virus-related cirrhosis. METHODS: Sixteen patients and ten healthy subjects were investigated. T lymphocytes were activated in vitro in the presence or absence of Vitamin D3 and then the proliferative response and IFN-γ and TNF-α production were assessed. RESULTS: Vitamin D3 potently reduced T-lymphocyte proliferation in a dose-related fashion. Similarly, FACS analysis and ELISA testing demonstrated that Vitamin D3 significantly decreased the response frequency and the response intensity of IFN-γ and TNF-α production in the whole CD3-positive T lymphocyte population as well as in "naive" CD4+ CD45RA+ and "memory" CD4+ CD45RO+ T lymphocyte subsets. The inhibitory effect of Vitamin D3 on T-cell proliferation and cytokine production was not different between patients and controls. No toxic effects were exerted by Vitamin D3 even at the higher concentration used (10nM). Finally, no statistically significant correlation was found between 25(OH)D serum levels and the proliferative response or cytokine production of T lymphocytes from transplanted patients. CONCLUSIONS: This study demonstrates that in patients transplanted for hepatitis C virus-related cirrhosis Vitamin D3 modulates T lymphocyte activation, and provides a rationale for the evaluation of this compound as an immunosuppressive agent in liver-transplanted patients.

Proinflammatory modulation of the surface and cytokine phenotype of monocytes in patients with acute Charcot foot.

OBJECTIVE: Despite increased information on the importance of an inappropriate inflammatory response in the acute Charcot process, there has been no previous attempt to define the specific pathways that mediate its pathogenesis. Here, the role played by monocytes was analyzed. RESEARCH DESIGN AND METHODS: The immune phenotype of peripheral monocytes was studied by fluorescence-activated cell sorter analysis comparing patients with acute Charcot (n = 10) in both the active and recovered phase, diabetic patients with neuropathy (with or without osteomyelitis), and normal control subjects. RESULTS: When compared with diabetic control subjects and healthy subjects, monocytes from acute Charcot patients showed a proinflammatory immune phenotype characterized by increased production of proinflammatory cytokines, reduced secretion of anti-inflammatory cytokines, increased expression of surface costimulatory molecules, and increased resistance to serum withdrawal-induced apoptosis. In addition, the pattern of circulating cytokines confirmed activation of proinflammatory cytokines. No modulation of the monocyte phenotype was documented in diabetic control subjects and healthy subjects, thus indicating that the proinflammatory alterations of monocytes are specific and causative of acute Charcot. CONCLUSIONS: Together, these data provide evidence for the role of proinflammatory changes in the immune phenotype of monocytes in the pathogenesis of acute Charcot. These alterations may explain the abnormally intense and prolonged inflammatory response that characterizes this disorder and may represent a potential therapeutic target for specific pharmacological interventions.

1Alpha,25-dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in human monocytes.

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1alpha,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-gamma but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.

Downregulation of CD40 ligand response in monocytes from sepsis patients.

It has been suggested that a defective adaptive immune response contributes to septic immunosuppression. Here, the response of monocytes to CD40 ligand (CD40L) for patients with sepsis due to infection with gram-negative organisms has been analyzed. Compared to cells from controls, monocytes from septic patients showed significantly reduced production of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-12 and were unable to acquire high levels of CD80 and CD86 molecules. These alterations were observed at the onset of sepsis and persisted at day 7. However, the ability of monocytes to respond to CD40L stimulation was partially but significantly restored in cells from patients who recovered from sepsis. In addition, costimulation of autologous CD4+ T lymphocytes by CD40L-activated monocytes from septic patients failed to induce cell proliferation and gamma interferon production. Finally, the ability of CD40L to rescue monocytes from apoptosis was severely impaired. We conclude that downregulation of the CD40L response may be an appropriate model for the monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.

Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: role of two halogen atoms at the indole ring in developing new analogues with improved antiviral activity.

Indolyl aryl sulfones bearing the 4,5-difluoro (10) or 5-chloro-4-fluoro (16) substitution pattern at the indole ring were potent inhibitors of HIV-1 WT and the NNRTI-resistant strains Y181C and K103N-Y181C. These compounds were highly effective against the 112 and the AB1 strains in lymphocytes and inhibited at nanomolar concentration the multiplication of the IIIBBa-L strain in macrophages. Compound 16 was exceptionally potent against RT WT and RTs carrying the K103N, Y181I, and L100I mutations.

Lipopolysaccharide desensitizes monocytes-macrophages to CD40 ligand stimulation.

Polymicrobial sepsis induces the suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. Here, we examined whether macrophages were refractory to only LPS or if they were unable to respond to other stimuli such as CD40 ligand (CD40L). Monocytic cells exposed in vitro to LPS showed a dose-dependent reduction of their ability to produce interleukin-12 and tumour necrosis factor-alpha upon subsequent CD40L stimulation, as compared to cells stimulated with CD40L alone. Similarly, LPS interfered with the up-regulation of CD40, CD80 and CD86 induced by CD40L in monocytic cells. The effect of LPS on the response of monocytes to CD40L was similar whether these cells were directly exposed to LPS or cocultured with LPS-pretreated cells, indicating that soluble factors released by LPS stimulation could mediate tolerance to CD40L. We also show that the functional alterations induced by LPS in monocytes can be reversed by indomethacin, thus suggesting a role for inducible cyclooxygenase in mediating the LPS-induced hyporesponsive state of monocytes to CD40L. In conclusion, we propose that in vitro CD40L tolerance may be an appropriate model of monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.

Indolyl aryl sulphones as HIV-1 non-nucleoside reverse transcriptase inhibitors: synthesis, biological evaluation and binding mode studies of new derivatives at indole-2-carboxamide.

New non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are active against the commonly occurring mutations of HIV are urgently needed for the treatment of AIDS. We synthesized new NNRTIs of the indolyl aryl sulphone (IAS) family, which are endowed with high antiviral potency against HIV-1 wt (wild-type), and the Y181C and K103N-Y181C drug resistant mutant strains. Several new compounds were highly active in lymphocytes infected with primary isolates carrying the K103N-V1081-M184V and L1001-V1081 mutations. The design of new IASs was based on three-dimensional quantitative structure-activity relationship (3D QSAR) studies and docking simulations. A cross-docking study was also undertaken to gain some insights in to the binding mode of the newly synthesized IASs in the wt and mutated isoforms of reverse transcriptase.

Design, molecular modeling, synthesis, and anti-HIV-1 activity of new indolyl aryl sulfones. Novel derivatives of the indole-2-carboxamide.

Molecular modeling studies and an updated highly predictive 3-D QSAR model led to the discovery of exceptionally potent indolyl aryl sulfones (IASs) characterized by the presence of either a pyrrolidyn-2-one nucleus at the indole-2-carboxamide or some substituents at the indole-2-carbohydrazide. Compounds 7 and 9 were found active in the sub-nanomolar range of concentration in both MT-4 and C8166 cell-based anti-HIV assays. These compounds, and in particular compound 9, also showed excellent inhibitory activity against both HIV-112 and HIV-AB1 primary isolates in lymphocytes and against HIV WT in macrophages.

Novel 1-[2-(diarylmethoxy)ethyl]-2-methyl-5-nitroimidazoles as HIV-1 non-nucleoside reverse transcriptase inhibitors. A structure-activity relationship investigation.

1-[2-(Diarylmethoxy)ethyl]-2-methyl-5-nitroimidazoles (DAMNIs) is a novel family of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) active at submicromolar concentration. Replacement of one phenyl ring of 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (4) with heterocyclic rings, such as 2-thienyl or 3-pyridinyl, led to novel DAMNIs with increased activity. In HIV-1 WT cell-based assay the racemic 1-{2-[alpha-(thiophen-2-yl)phenylmethoxy]ethyl}-2-methyl-5-nitroimidazole (7) (EC(50) = 0.03 microM) proved 5 times more active than compound 4. Docking experiments showed that the introduction of a chiral center would not affect the binding of both (R)-7 and (S)-7. The internal scoring function of the Autodock program calculated the same inhibition constant (K(i) = 7.9 nM) for the two enantiomers. Compounds 7 (ID(50) = 8.25 microM) were found more active than efavirenz (ID(50) = 25 microM) against the viral RT carrying the K103N mutation, suggesting for these compounds a potential use in efavirenz based anti-AIDS regimens.