Reduced levels of both circulating CD4+ CD25+ CD127(low/neg) and CD4+ CD8(neg) invariant natural killer regulatory T cells in stable heart transplant recipients.

A cross-regulation between two regulatory T cell (T(reg) ) subsets [CD4(+) CD25(+) and invariant natural killer (NK) T - iNK T] has been described to be important for allograft tolerance induction. However, few studies have evaluated these cellular subsets in stable recipients as correlates of favourable clinical outcome after heart transplantation. T(reg) and iNK T cell levels were assayed by flow cytometry in peripheral blood samples from 44 heart transplant recipients at a 2-year interval in 38 patients, and related to clinical outcome. Multi-parameter flow cytometry used CD4/CD25/CD127 labelling to best identify T(reg) , and a standard CD3/CD4/CD8/Vα24/Vß11 labelling strategy to appreciate the proportions of iNK T cells. Both subtypes of potentially tolerogenic cells were found to be decreased in stable heart transplant recipients, with similar or further decreased levels after 2 years. Interestingly, the patient who presented with several rejection-suggesting incidents over this period displayed a greater than twofold increase of both cell subsets. These results suggest that CD4(+) CD25(+) CD127(low/neg) T(reg) and iNK T cells could be involved in the local control of organ rejection, by modulating immune responses in situ, in clinically stable patients. The measurement of these cell subsets in peripheral blood could be useful for non-invasive monitoring of heart transplant recipients, especially in the growing context of tolerance-induction trials.

Alteration of the immunological synapse in lung cancer: a microenvironmental approach.

This study was designed to investigate the immunological properties of stroma reaction T cells and tumoral cells by comparison with non-tumoral lung tissue and local lymph nodes in order to explore interactions between tumour cells and the immune system. Immunodetection of major histocompatibility complex (MHC) molecules, CD3/T cell receptor (TCR) complex and T cell subsets markers was carried out in situ on frozen sections, and the semi-quantitative expression of CD3, CD4 and CD8 was examined in flow cytometry on lymphocytes of nodal, tumoral and healthy lung tissue from 62 patients with non-small cell lung cancer. This study showed alterations on lymphocytes and tumour cells in lung cancer, consistent with an impairment of T cell activation. CD3, TCR alpha beta and accessory molecules expression is down-modulated on peri- or intra-tumoral lymphocytes. MHC class I and class II molecules are down-modulated significantly on tumour cells. Other differences were noted, such as the reversed CD4/CD8 ratio of tumour infiltrating cells, compared to healthy lung tissues, consistent with the development of cytotoxic anti-tumoral responses. This study reports on the presence of a strong in vivo immunomodulating effect of tumour cells in human non-small cell lung cancer, likely to impair proper formation of the immunological synapse.

Isoelectric restriction of human immunoglobulin isotypes.

The partition of human serum immunoglobulins along a pH gradient of ampholynes was investigated using the recently developed method of preparative isoelectrofocusing. Each isotype was demonstrated to display a specific pI range, with limited overlapping. IgA appear to be the most acidic serum immunoglobulins while IgG are clearly basic.

Back-pack mice as a model of renal mesangial IgA dimers deposition.

Mesangial IgA in IgA nephropathy are dimers with a J chain but no poly-Ig receptor. This molecular structure has led to the hypothesis that these IgA are issued from the lamina propria of mucosal areas, reaching the kidney by way of the peripheral blood. The availability of hybridomas producing IgA dimers provided an opportunity to test this hypothesis in a new experimental model of IgA nephropathy. Mice were injected subcutaneously (back-pack mice) or intraperitoneally with hybridoma cells secreting either monoclonal IgA dimers, or monoclonal IgA monomers. The influence of immune complex formation was also tested in both these models. Renal IgA deposition was investigated 12 days after the injection of hybridoma cells. Backpack mice developed highly vascularized subcutaneous tumors. Mesangial IgA deposits were observed only in dimeric IgA hybridoma back-pack animals. No significant staining was observed in glomeruli from animals injected with hybridoma cells producing monomeric IgA. None of the hybridomas induced mesangial deposition when injected intraperitoneally. This animal model demonstrates the capacity of circulating IgA dimers to spontaneously form mesangial deposits and contributes to confirm the involvement of abnormalities of mucosal immunity in the pathogenesis of IgA nephropathy.

Alternative rearrangements of immunoglobulin light chain genes in human leukemia.

Immunoglobulin heavy and light chain genes undergo rearrangement before they can be expressed by B-cells. A sequence of successive rearrangements has been proposed, suggesting that these events are initiated on heavy chain genes and are followed by sequential attempts of light chain gene rearrangements involving kappa gene alleles before lambda genes. This hypothesis, mainly established on B-cell clones of medullary origin, is consistent with the predominance of kappa chains among human serum immunoglobulins. However, data from tumors or normal lymphoid tissue suggest that lambda chain expression could be favored outside the bone marrow, due to an alternative rearrangement hierarchy. We investigated this hypothesis further on 17 leukemia samples. In three instances, we observed that lambda genes had undergone rearrangement while kappa genes remained in germline configuration. These data support the hypothesis of occasional alternative rearrangements of light chain genes.

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

[Ovarian autoimmunity and ovarian pathologies: antigenic targets and diagnostic significance]. / Auto-immunité anti-ovarienne et pathologies ovariennes: cibles antigéniques et implications diagnostiques.

The involvement of serum anti-ovarian autoantibodies (AOA) in ovarian pathology still remains controversial. In some cases of clinically patent ovarian failure, there seems to be a causal relationship between AOA and the ovarian disease. In patients with various organ-specific or systemic autoimmune diseases, or with unexplained, repeated reproductive failure, but otherwise normal ovarian function, it is even more difficult to determine the significance of AOA for several reasons: i) AOA recognize many different antigenic targets in the ovary ii) the antiovarian response may be transient or variable with time iii) the presence of AOA does not imply their aetiopathogenic role in the disease. The present paper reviews the clinical significance of AOA based on their ovarian targets as far as they have been identified until now.

LFA1 expression in HIV infection.

The expression of the adhesion molecule LFA1 was investigated in mononuclear cells from 200 samples of peripheral blood obtained from asymptomatic HIV-infected individuals and AIDS patients. The numbers and percentages of LFAI-positive cells were established by indirect immunofluorescence. A significant decrease in the number of labelled cells was observed (mean percentage 68.9) while 100% of the cells were positive in controls. These data suggest that LFA1-mediated cell-cell cooperation might be impaired in HIV infection, adding to the immune deficiency related to the disappearance of CD4+ cells.

A CD4-like molecule can be expressed in vivo in human parathyroid.

The expression of several epitopes of CD4, a molecule usually restricted to a subset of T-lymphocytes, was observed after immunofluorescent labeling of frozen-cut sections of human parathyroid. Seven samples obtained from three subjects presenting adenomas and from seven renal insufficiency patients with secondary or tertiary hyperplasia were found to express this molecule. Dot enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and Western blotting were used to characterize this peptide in cytosol and membrane fractions of these glands. These studies confirmed, in the parathyroids found positive in immunofluorescence, the presence of a protein with a mol wt similar to that of lymphocyte-derived CD4.

Monocyte-derived IL-10-secreting dendritic cells in choroid plexus epithelium.

Choroid plexuses form an interface between peripheral blood and cerebrospinal fluid. Dendritic-like cells have been reported in a few studies of choroid plexuses in man. Here we used electron microscopy and immunophenotyping to precise the morphologic features and phenotype of these cells. Examination of 10 human choroid plexuses evidenced intra-epithelial dendritic cells with a clear cytoplasm, reniform nucleus and long expansions. These cells express MHC Class II, CD11b, CD14, CD32, CD68 and IL-10, but not CD40, CD80 or CD86, suggesting an immunosuppressive role for these dendritic cells. Their sentinel position could make them participate to the immunological silence of the brain.

Anti-myelin oligodendrocyte glycoprotein B-cell responses in multiple sclerosis.

Humoral auto-immunity to the myelin oligodendrocyte glycoprotein (MOG) is likely involved in the pathogenesis of multiple sclerosis (MS). In 44 MS patients and 30 controls, Ig-producing B cells were identified by their isotype and as MOG-specific spot-forming cells (SFC). Peripheral anti-MOG antibodies were assayed in ELISA as well as anti-butyrophilin antibodies to investigate for molecular mimicry. MS patients had significantly higher levels of IgA- and MOG-SFC than controls, as well as significantly higher antibody responses to MOG and butyrophilin. These data provide added support for the implication of anti-MOG humoral immunity in the pathophysiology of MS, and suggest a balance of systemic (anti-self) and mucosal (environment-modulated) immune reactions in an attempt at regulating the pathogenic specific immune response.

Increased usage of TCR V-beta8 in kidney transplant recipients with aberrant immune reconstitution and clinical complications.

BACKGROUND: The efficiency of immunosuppressive drugs prescribed after organ transplantation is mostly monitored through clinical and biological signs of organ rejection or infection. However, it may be expected that some patients develop subtle alterations of their reconstituting immune system, not immediately associated with clinical events. Identification of such anomalies could be useful to alert clinicians for possible future complications. METHODS: A systematic follow-up of peripheral lymphocyte subsets, performed in a cohort of 89 kidney transplant recipients, identified severely skewed CD4/CD8 ratios in 32 patients. For 19 patients, the expression of specific T cell receptor fragments was examined using a panel of 10 monoclonal antibodies. Abnormal control of spontaneously Epstein Barr virus-infected B cells was tested by investigating for the generation of spontaneous lymphoblastoid cell lines in 17 cases. The incidence of rejection and infectious episodes was monitored. RESULTS: A bias in T cell receptor fragments usage was detected in 14/19 cases, involving Vbeta8 in all cases. Spontaneous lymphoblastoid cell lines of Epstein Barr positive B blasts developed in 9 of 17 cases. Eleven patients had early rejection episodes and 16 presented with viral primo-infection or reactivation. The incidence of rejection and infectious episodes was higher in the group of 32 patients who developed such abnormal patterns than in the 57 who did not. CONCLUSION: Transient bias in the T cell receptor repertoire may be observed during immune reconstitution after kidney transplantation, perhaps related to abnormal lymphocyte functions and associated to an impaired control of rejection and/or infectious agents.

Early increase of peripheral B cell levels in kidney transplant recipients with CMV infection or reactivation.

BACKGROUND: Cytomegalovirus (CMV) infection or reactivation is a frequent complication of renal transplantation. Diagnosis of these conditions relies on the detection of circulating antigen, or of specific IgM and/or IgG, which develop over several weeks. Evocative clinical features may be detected earlier, but lack specificity. Rapid and early changes in the partition of lymphocyte subsets could be an additional indication of pending CMV infection. METHODS: A systematic follow-up of peripheral B lymphocytes identified immunophenotypically by the determination of surface immunoglobulins (sIg), performed in 97 kidney transplant recipients, allowed to identify transient increases apparently predictive of CMV primo-infection or reactivation over the next 3 months. To better define the nature of these B cells, an extended investigation was performed for 14 prospective patients. In addition to surface Ig, membrane CD19, HLA-DR, and CD80 expression were explored. The cytoplasmic presence of mu, kappa, and lambda chains was also examined. B cell function was investigated using the ELISPOT technique, which allows an enumeration of the populations of IgG, IgA, and IgM secreting B cells. RESULTS: Retrospective analysis of the clinical outcome of the cohort of 97 patients evidenced that early transient increases in B cell levels were significantly (P<0.0001) associated with CMV infection. The same trend was noted in the smaller series of patients who benefited from a more extensive investigation of B cells, 10 of whom presented clinical or biological signs of CMV infection. Mature B cells, expressing surface Ig, CD19, DR, and CD80 are those presenting transient increases. No significant variation of preB (cmu+/kappalambda-) or activated (spot-forming) cells was evidenced in these patients. CONCLUSION: Individual examination of each patient's immune reconstitution profile allows to detect transient peaks of mature B cell during the initial immunosuppressive therapy, that appear to be predictive of oncoming CMV infection or reactivation.

From Peyer's patches to tonsils. Specific stimulation with ribosomal immunotherapy.

Ribosomal immunotherapy has been successfully used since the 1960s to boost the immune system and provide protection against microbial infections. We have investigated both whether and how these immunostimulants behave as natural immunogens in the mucosa-associated immune system. According to current understanding of the physiology of the mucosal immune response, intestinal Peyer's patches and the related solitary nodules are the primary inductive sites involved in the immune protection of all mucosal surfaces. Sensitised lymphocytes generated at these sites reach the general circulation through lymphatic drainage and relocate in mucosal areas by means of specialised 'high endothelial venules'. We hypothesised that orally administered ribosomal preparations would yield sensitised B cells specific for bacterial antigens from the parent strains. These cells should then be detectable in the peripheral blood after ribosomal intake, and identifiable as plasma cells in mucosae-associated tissues after completing their terminal differentiation. Ultimately, specific IgA should appear in secretions. To this end, we studied the immune responses generated in children and adults after 'Ribomunyl' administration, according to various consecutive protocols. The initial hypothesis was confirmed by the identification of specific B cells in the peripheral blood, plasma cells in the tonsillar tissue and specific IgA in the saliva. An animal model involving the use of twin sheep enabled detection of the specific cells in mesenteric and cervical lymph nodes. Analysis of these data indicates that ribosomal preparations trigger the production of lymphocytes specific for both ribosomes themselves and whole bacterial antigens. This supports the fact that small antigenic motifs are carried as partly synthesised peptides on the ribosomal particles. Therefore, ribosomes boost an array of B cells that are specific for many antigenic determinants of the bacteria from which they are extracted. We were also able to show that the stimulation provided was specific, since no response to other bacteria could be detected. Finally, analysis of the kinetics of this stimulation confirmed that oral immunisation generates rapid and transient secretory responses, building increased numbers of memory cells that are readily available to respond to further challenges by either more ribosomal preparations or potential pathogens.

Comparative immunohistochemical characteristics of human choroid plexus in vascular and Alzheimer's dementia.

Autoimmune alterations are indirectly supported in Alzheimer's disease by the demonstration of circulating antibodies directed to the epithelial basement membrane (BM) of the choroid plexus. We used immunohistochemical methods to compare the characteristics of choroid plexuses obtained postmortem from 15 patients. Six had a diagnosis of Alzheimer's disease, five had multi-infarct dementia (MID), and one suffered from mixed dementia. Similar tissue from three age-matched, non-demented controls was studied as well. Age-related psammoma bodies, lipofucsin, and flattened epithelial cells were present in all cases. Specific alterations were evident in Alzheimer's disease patients only. These were comprised of pseudolinear deposits of immunoglobulin (Ig)G and coarse deposits of C1q along the thickened and segmented epithelial BM, and were associated with IgM in five cases. Although no lymphoid infiltration was demonstrated, MHC Class II+ macrophages were observed in the plexus stroma, and numerous epithelial cells were class II+. These observations suggest that immune alterations, possibly of autoimmune origin, may be involved in Alzheimer's disease, leading to severe lesions of the choroid plexus. Such anomalies could be responsible for some of the alterations of cerebrospinal fluid (CSF) production or composition noted in this disease.

Synthetic extracellular matrix supports healing of mucogingival surgery donor sites.

Palatal donor sites are commonly used in periodontal surgery. Their healing speed determines the patient's comfort. This may be enhanced by the use of collagen sponges, supplemented with various components of the extracellular matrix, which display chemotactic properties and enhance the proliferation and synthesis activity of fibroblasts. A collagen-based dressing supplemented with such extracellular matrix components, including chondroitin 4 sulfate, heparan sulfate, and fibronectin was used, in an attempt to facilitate the healing of donor sites in 10 periodontal patients. Immunohistological techniques were used on biopsy samples from the margin of these volunteer's healing donor sites to appreciate tissue reconstruction around the synthetic material. Our results indicate a fast epithelial growth, neovascularization, and spatial organization of the new matrix. Limited and topographically selective inflammatory reactions, characterized by polymorphonuclears and mononuclear cells seen near the wound's margin, could be responsible for the production of soluble factors supporting reconstruction.

Plasma cells producing lambda light chains are predominant in human gut and tonsils. An immunohistomorphometric study.

An immunohistomorphometric study was performed on human samples of duodenum (10) and tonsil (25) to assess the numbers of plasma cells producing kappa or lambda chains. Different reagents were used and carefully assayed for specificity and absence of cross-reactivity. Kappa chains were found predominantly with these antibodies in 46 bone marrow-derived B-cell proliferations used as reagents' control samples. In contrast, plasma cells producing lambda chains were found to be more numerous in the samples of mucosal tissues. The kappa/lambda ratio observed was 0.53. This finding could be another feature reflecting the autonomy of the immune system of mucosae (MALT) in humans.

IgA nephropathy and alcoholic liver cirrhosis. A prospective necropsy study.

The incidence of mesangial IgA nephropathy (mIgAN) was investigated in a series of patients with alcoholic liver cirrhosis (ALC). Biologic parameters classically reported in IgAN were assessed in 98 patients, namely hematuria, proteinuria, and serum IgA. An immunohistologic study of the liver and kidney was performed in 33 patients who died during the study. Renal data were compared with those obtained in a matched necropsic series of controls. This study confirmed a global elevation of serum IgA levels in ALC. A possible hepatic origin of these immunoglobulins was supported by the observation of plasma cells in portal spaces in 68% of the patients. Biologic signs of renal disease consistent with mIgAN were observed in 16% of the patients; IgAN was diagnosed in 18% of patients with ALC and 10% of the controls. These data suggest that the incidence of mIgAN in ALC is not different than in the general population.

Automated cell count in flow cytometry: a valuable tool to assess CD4 absolute levels in peripheral blood.

The enumeration of lymphocyte subsets in absolute counts has long relied on different methods applied separately to whole blood cell count, lymphocyte differential appreciation, and flow cytometric evaluation of lymphocyte subsets percentages. The development of multicolor labeling methods inflow cytometry now allows a more homogeneous appreciation of several cell subsets among gated lymphocytes. The use of internal calibrators, such as microbead suspensions, also permits a direct appreciation of subsets in absolute counts in a single-platform method. These methods were compared with a traditional multiplatform method of assessing absolute counts of lymphocyte subsets in a pilot study in which all manipulations were performed by 1 person and in a full-scale larger study performed in the normal working conditions of a hospital laboratory. Microspheres seem to be a reliable tool to perform absolute count enumeration inflow cytometry, but several precautions in the sample preparation and flow cytometric analysis are required.

Antibodies to choroid plexus in senile dementia of Alzheimer's type.

AIMS: To investigate whether autoantibodies to choroid plexus are present in human senile dementia. METHODS: Serum samples from 40 elderly people presenting with characteristic, diagnostic criteria of senile dementia of Alzheimer's type and 20 age matched healthy controls were tested by indirect immunofluorescence for the presence of autoantibodies to choroid plexuses, using frozen sections of rat or human fetal brain tissue. RESULTS: Significant labelling of choroid plexus basement membrane was observed in 17 of the 40 samples from patients with senile dementia; in the control series one sample of rat but not human plexus labelled positively (p < 0.01). CONCLUSIONS: The antibodies identified in this series of patients with Alzheimer's disease suggest that autoimmune mechanisms might be responsible for some of the changes in cerebrospinal fluid production described in this disorder.