Long-term administration of IgG2a anti-NK1.1 monoclonal antibody ameliorates lupus-like disease in NZB/W mice in spite of an early worsening induced by an IgG2a-dependent BAFF/BLyS production

The role of natural killer (NK) T cells in the development of lupus-like disease in mice is still controversial. We treated NZB/W mice with anti-NK1.1 monoclonal antibodies (mAbs) and our results revealed that administration of either an irrelevant immunoglobulin G2a (IgG2a) mAb or an IgG2a anti-NK1.1 mAb increased the production of anti-dsDNA antibodies in young NZB/W mice. However, the continuous administration of an anti-NK1.1 mAb protected aged NZB/W mice from glomerular injury, leading to prolonged survival and stabilization of the proteinuria. Conversely, the administration of the control IgG2a mAb led to an aggravation of the lupus-like disease. Augmented titres of anti-dsDNA in NZB/W mice, upon IgG2a administration, correlated with the production of BAFF/BLyS by dendritic, B and T cells. Treatment with an anti-NK1.1 mAb reduced the levels of interleukin-16, produced by T cells, in spleen cell culture supernatants from aged NZB/W. Adoptive transfer of NK T cells from aged to young NZB/W accelerated the production of anti-dsDNA in recipient NZB/W mice, suggesting that NK T cells from aged NZB/W are endowed with a B-cell helper activity. In vitro studies, using purified NK T cells from aged NZB/W, showed that these cells provided helper B-cell activity for the production of anti-dsDNA. We concluded that NK T cells are involved in the progression of lupus-like disease in mature NZB/W mice and that immunoglobulin of the IgG2a isotype has an enhancing effect on antibody synthesis due to the induction of BAFF/BLyS, and therefore have a deleterious effect in the NZB/W mouse physiology.

Anti-Group A Streptococcal Vaccine Epitope STRUCTURE, STABILITY, AND ITS ABILITY TO INTERACT WITH HLA CLASS II MOLECULES

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue alpha-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 alpha-helix residues, whereas the B cell epitope is in the second microdomain and showed no alpha-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCoroverlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF)T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Expression of constitutively activated human c-Kit in myb transformed early myeloid cells leads to factor independence, histiocytic differentiation, and tumorigenicity

The cDNAs encoding wild type (WT) human receptor tyrosine kinase c-Kit and a constitutively activated mutant, V816Kit, were introduced into granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent early murine hemopoietic cells, which had been transformed with activated Myb, WTKit cells were able to grow in the presence of the human ligand for Kit, stem cell factor (SCF), but displayed reduced growth and clonogenic potential in either SCF or GM-CSF compared with the parental cells in GM-CSF. In contrast, V816Kit cells grew without factor at a higher rate than the parental cells in GM-CSF and displayed increased clonogenicity. Dissection of the growth characteristics in liquid culture showed that in the presence of appropriate factors, the different populations had similar proliferation rates, but that V816Kit profoundly increased cell survival compared with WTKit or parental cells, This suggests that the signals transduced by WTKit activated with SCF, and by V816Kit, were not identical. Also, WTKit and V816Kit-expressing cells both varied from the early myeloid progenitor phenotype of the parental cells and gave rise to a small number of large to giant adherent cells that expressed macrophage (alpha-naphthyl acetate) esterase and neutrophil (naphtol-AS-D-chloroacetate) esterase, were highly phagocytic and phenotypically resembled histiocytes. Thus, WTKit activated by SCF and V816Kit were able to induce differentiation in a proportion of Myb-transformed myeloid cells. The factor independent V816Kit cells, unlike the parental and WTKit expressing cells, were shown to produce tumors of highly mitotic, invasive cells at various stages of differentiation in syngeneic mice. These results imply that constitutively activated Kit can promote the development of differentiated myeloid tumors and that its oncogenic effects are not restricted to lineages (mast cell and B-cell acute lymphoblastic leukemia), which have been reported previously. Furthermore, the mixed populations of cells in culture and in the tumors phenotypically resembled the leukemic cells from patients with monocytic leukemia with histiocytic differentiation (acute myeloid leukemia-M5c), a newly proposed subtype of myeloid leukemia. (C) 1997 by The American Society of Hematology.

The spectrum of autoantibodies in IPEX syndrome is broad and includes anti-mitochondrial autoantibodies

IPEX syndrome is a congenital disorder of immune regulation caused by mutations in the FOXP3 gene, which is required for the suppressive function of naturally arising CD4 + CD25 + regulatory T cells. In this case series we evaluated serum samples from 12 patients with IPEX syndrome for the presence of common autoantibodies associated with a broad range of autoimmune disorders. We note that 75% of patients (9/12) had 1 or more autoantibodies, an incidence far above the cumulative rate observed in the general population. The range of autoantibodies differed between patients and there was no predominant autoantibody or pattern of autoantibodies present in this cohort. Surprisingly, one patient had high-titer anti-mitochondrial antibodies (AMA) typically associated with primary biliary cirrhosis (PBC) although the patient had no signs of cholestasis. PBC is a well-characterized autoimmune disease that occurs primarily in women and includes the serological hallmarks of serum AMA and elevated IgM which were both present in this patient. PBC is virtually absent in children with the exception of one reported child with interleukin 2 receptor a (CD25) deficiency which is associated with an IPEX-like regulatory T cell dysfunction. Based on the present data and the available literature we suggest a direct role for CD4 + CD25 + regulatory T cells in restraining B cell autoantibody production and that defects in regulatory T cells may be crucial to the development of PBC. (C) 2010 Elsevier Ltd. All rights reserved.

Autoimmunity in Hyper-IgM syndrome

Introduction Immunodeficiency with hyper-IgM (HIGM) results from genetic defects in the CD40-CD40 ligand (CD40L) pathway or in the enzymes required for immunoglobulin class switch recombination and somatic hypermutation. HIGM can thus be associated with an impairment of both B-cell and T-cell activation. Results and discussions There are seven main subtypes of HIGM and the most frequent is X-linked HIGM, resulting from CD40L mutations. In addition to the susceptibility to recurrent and opportunistic infections, these patients are prone to autoimmune manifestations, especially hemato-logic abnormalities, arthritis, and inflammatory bowel disease. Furthermore, organ-specific autoantibodies are commonly found in HIGM patients. Conclusions The mechanisms by which HIGM associates to autoimmunity are not completely elucidated but a defective development of regulatory T cells, the presence of IgM autoantibodies and an impaired peripheral B-cell tolerance checkpoint have been implicated. This article reviews the main subtypes of HIGM syndrome, the clinical autoinumme manifestations found in these patients, and the possible mechanisms that would explain this association.

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-gamma- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response. (C) 2010 Elsevier GmbH. All rights reserved.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric IAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory. (C) 2010 Elsevier Inc. All rights reserved.

C4d staining in post-reperfusion renal biopsy is not useful for the early detection of antibody-mediated rejection when CDC crossmatching is negative

Background. Sensitized patients (pts) may develop acute antibody-mediated rejection (AMR) due to preformed donor-specific antibodies, undetected by pre-transplant complement-dependent cytotoxicity (CDC) crossmatch (XM). We hypothesized that C4d staining in 1-h post-reperfusion biopsies (1-h Bx) could detect early complement activation in the renal allograft due to preformed donor-specific antibodies. Methods. To test this hypothesis, renal transplants (n = 229) performed between June 2005 and December 2007 were entered into a prospective study of 1-h Bx and stained for C4d by immunofluorescence. Transplants were performed against a negative T-cell CDC-XM with the exception of three cases with a positive B-cell XM. Results. All 229 1-h Bx stained negative for C4d. Fourteen pts (6%) developed AMR. None of the 14 protocol 1-h Bx stained positive for C4d in peritubular capillaries (PTC). However, all indication biopsies-that diagnosed AMR-performed at a median of 8 days after transplantation stained for C4d in PTC. Conclusions. These data show that C4d staining in 1-h Bx is, in general, not useful for the early detection of AMR when CDC-XM is negative.

Survival and Recurrence in Nonmycosis Fungoides Primary Cutaneous Lymphoma

Purpose: To evaluate overall and relapse-free survival (RFS) in patients with nonmycosis fungoides (non-MF) primary cutaneous lymphoma (PCL). Methods: Thirty-eight patients with PCL excluding cases of MF treated between 1993 and 2006 were analyzed retrospectively. Survival statistics were estimated by the methods of Kaplan and Meier, and univariate and multivariate significance testing were performed by Cox regression analysis. Results: The median follow-up was 34.6 months (range, 2-138.3 months). The overall survival for the entire study population, at 5 and 10 years, was 97% and 78%, respectively. The RFS for the entire study population, at 5 and 10 years, was 30% and 22%, respectively. For those who received radiotherapy (RT) as a component of their initial therapy, the RFS at 5 and 10 years was 48% and 36%, respectively. Among those receiving RT who relapsed, the site of relapse was out-of-field in 82% of the cases. In our multivariate analysis, only RT as a component of the initial therapy and the absence of bulky disease had a statistically significant improvement in RFS (P = 0.01 and < 0.01, respectively). Conclusion: RT improves the local control and RFS of patients with non-MF PCL.

Burkitt Lymphoma in Brazil Is Characterized by Geographically Distinct Clinicopathologic Features

Burkitt lymphoma (BL) is a highly aggressive non-Hodgkin lymphoma with a consistent MYC translocation. Epstein-Barr virus (EBV) has been associated with BL at different frequencies, depending on the clinical variant and geographic regions. This is a large-scale study of BL in Brazil, including 234 patients from 5 geographic regions that are widely disparate socioeconomically, including pediatric (61.1%) and adult (37.6%) populations. EBV was present in 52.6% of all BL cases, varying from 29% (12/42) in the South to 76% (13/17) in the North. Most of the cases were EBV type A. The frequency was higher in the pediatric group, and EBV association within this age range predominated in all regions except the South. Expression of p53 protein was observed inn 16.2%, and only rare cases showed p63 expression. BL in Brazil is regionally distinct and has a low incidence of p53 overexpression and a higher-than-expected association with EBV in sporadic cases.

Change in expression of miR-let7c, miR-100, and miR-218 from high grade localized prostate cancer to metastasis

Objective: MicroRNAs (miRNAs) are small noncoding regulatory RNAs (19-25 nucleotides) that play a major role in regulation of gene expression. They are responsible for the control of fundamental cellular processes that has been reported to be involved in human tumorigenesis. The characterization of miRNA profiles in human tumors is crucial for the understanding of carcinogenesis processes, finding of new tumor markers, and discovering of specific targets for the development of innovative therapies. The aim of this study is to find miRNAs involved in prostate cancer progression comparing the profile of miRNA expressed by localized high grade carcinoma and bone metastasis. Material and methods: Two groups of tumors where submitted to analyses. The first is characterized by 18 patients who underwent radical prostatectomy for treatment of localized high grade prostate carcinoma (PC) with mean Gleason score 8.6, all staged pT3. The second group is composed of 4 patients with metastatic, androgen-independent prostate carcinoma, and 2 PC cell lines. LNCaP derived from a metastatic PC to a lymph node, and another derived from an obstructive, androgen-independent PC (PcBRA1). Expression analysis of 14 miRNAs was carried out using quantitative RT-PCR. Results: miR-let7c, miR-100, and miR-218 were significantly overexpressed by all localized high GS, pT3 PC in comparison with metastatic carcinoma. (35.065 vs. 0.996 P < 0.001), (55.550 vs. 8.314, P = 0.010), and (33.549 vs. 2.748, P = 0.001), respectively. Conclusion: We hypothesize that miR-let7c, miR-100, and miR-218 may be involved in the process of metastasization of PC, and their role as controllers of the expression of RAS, c-myc, Laminin 5 beta 3, THAP2, SMARCA5, and BAZ2A should be matter of additional studies. (C) 2011 Elsevier Inc. All rights reserved.

Low mRNA Expression of the Apoptosis-Related Genes CASP3, CASP8, and FAS Is Associated With Low Induction Treatment Response in Childhood Acute Lymphoblastic Leukemia (ALL)

Background. Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. Results. A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P<0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P=0.01) and low risk group classification (P=0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P=0.03, P=0.02 and P=0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P=0.02). Conclusion. These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL. Pediatr Blood Cancer 2010;55:100-107. (C) 2010 Wiley-Liss, Inc.

Peroxiredoxin I is differentially expressed in multiple myelomas and in plasmablastic lymphomas

BACKGROUND: Plasmablastic lymphoma (PBL) and multiple myeloma (MM) are B cell-derived malignancies that share many morphologic and immunophenotypic traits, making the differential diagnosis particularly complicated. We have recently demonstrated that peroxiredoxin I (PrdxI) is expressed in plasma cells but not in B lymphocytes, suggesting that its expression is development-associated. AIM: To analyze PrdxI expression in PBL and in MM in order to study its utilization as an additional diagnostic molecular tool. METHODS AND RESULTS: Eight cases of PBL and nine of MM were studied by immunohistochemistry. We have demonstrated that PrdxI expression is closely connected with the immunoglobulin production capacity of the cells, which means high in MM, but absent in PBL cases, except one, wherein few cells were stained. CONCLUSIONS: We hypothesize PrdxI as a component of the unfolded protein response (UPR), an adaptive pathway essential for plasma cell differentiation. As we have not detected immunoglobulin in our PBL cases, we suggest that UPR was not activated in the cells, accounting for the impediment of the developmental process, and for the inhibition of PrdxI expression observed. PrdxI could be considered an additional plasma cell functional marker and could also be speculated as a therapeutic target in the treatment of MM. Oral Diseases (2008) 14, 741-746

Costimulatory molecules in human periodontal disease tissue

An immunoperoxidase technique was used to examine CD28, CD152, CD80 and CD86 positive cells in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups (small, intermediate, large) according to the size of the infiltrate. The percent CD28+ T cells in the connective tissue infiltrates was highly variable with no differences between the healthy/gingivitis and periodontitis groups. While there was an increase in positive cells in intermediate infiltrates from both healthy/gingivitis (28.5%) and periodontitis (21.4%) patients compared with small infiltrates (8.6% and 11.8%, respectively), this was not significant, although the percent CD28+ T cells did increase significantly in tissues with increased proportions of B cells relative to T cells (p=0.047). A mean of less than 5% infiltrating T cells were CD152+ which was significantly lower than the mean percent CD28+ T cells in intermediate healthy/gingivitis lesions (p=0.021). The mean percent CD80+ and CD86+ B cells and macrophages was 1–7% and 8–16%, respectively, the difference being significant in intermediate healthy/gingivitis tissues (p=0.012). Analysis of these cells in relation to increasing numbers of B cells in proportion to T cells and also to macrophages, suggested that CD80 was expressed predominantly by macrophages while CD86 was expressed by both macrophages and B cells. Few endothelial cells expressed CD80 or CD86. Keratinocytes displayed cytoplasmic staining of CD80 rather than CD86 although the numbers of positive specimens in the healthy/gingivitis and periodontitis groups reduced with increasing inflammation. In conclusion, percentages of CD28, CD152, CD80 and CD86 did not reflect differences in clinical status. However, the percent CD28+ T cells increased with increasing size of infiltrate and with increasing proportions of B cells suggesting increased T/B cell interactions with increasing inflammation. The percent CD152+ cells remained low indicating that CD152 may not be involved in negative regulation of T cells in periodontal disease. CD80 and CD86 have been reported to promote Th1 and Th2 responses, respectively, and the higher percent CD86+ cells suggests a predominance of Th2 responses in both healthy/gingivitis and periodontitis tissues. Nevertheless, other factors including cytokines themselves and chemokines which modulate T cell cytokine profiles must be monitored to determine the nature of Th1/Th2 responses in periodontal disease.