CD4-positive Lymphocytes Research Articles

B7-H4 (DD-O110) is overexpressed in high risk uterine endometrioid adenocarcinomas and inversely correlated with tumor T-cell infiltration

Objectives and methods B7-H4 (DD-O110), a member of the B7 family, negatively regulates T cell-mediated immune response. Previous studies have shown that B7-H4 is highly expressed in endometrioid ovarian cancers with relatively low levels of expression in normal ovary which was confirmed by Western blot. The present study was designed to localize B7-H4 expression by immunohistochemistry (IHC) in normal endometrium, endometrial hyperplasia and uterine endometrioid adenocarcinoma. The pattern of B7-H4 localization was compared with the IHC detection of CD3 and CD8-positive T lymphocytes and CD14 positive macrophages to investigate the role of B7-H4 in the regulation of tumor immune surveillance. B7-H4 expression was evaluated in apoptotic tumor cells. Results The proportion and intensity of B7-H4 staining were increased in the progression from normal, hyperplastic and malignant endometrial glandular mucosa. B7-H4 showed a predominantly apical membranous staining (pattern 1) in normal and hyperplastic endometrial epithelium but showed intense circumferential membranous and cytoplasmic staining (pattern 2) in a majority of endometrioid carcinoma cases ( p = 0.018). The proportion of B7-H4 positive tumor cells and staining intensity was also higher in high risk tumors than in low risk tumors ( p = 0.001 and p = 0.032, respectively). The proportion of B7-H4 positive tumor cells was inversely related to the number of CD3-positive and CD8-positive tumor-associated lymphocytes (TALs). There was a positive correlation between B7-H4 pattern 2 staining and both CD3-positive and CD8-positive tumor-infiltrating lymphocytes (TILs) ( p = 0.039 and p = 0.031, respectively). Conclusions B7-H4 is overexpressed in hyperplastic and malignant endometrial epithelium and is correlated with the number T cells associated with the tumor. These results suggest that B7-H4 overexpression may reflect a more aggressive biologic potential and may play a role in tumor immune surveillance mechanisms.

Severe Human Lower Respiratory Tract Illness Caused by Respiratory Syncytial Virus and Influenza Virus Is Characterized by the Absence of Pulmonary Cytotoxic Lymphocyte Responses

Background. Respiratory syncytial virus (RSV) and influenza virus are common causes of infantile lower respiratory tract infection (LRTI). It is widely believed that both viral replication and inappropriately enhanced immune responses contribute to disease severity. In infants, RSV LRTI is known to be more severe than influenza virus LRTI. Methods. We compared cytokines and chemokines in secretions of infants surviving various forms of respiratory illness caused by RSV or influenza viruses, to determine which mediators were associated with more-severe illness. We analyzed lung tissue from infants with fatal cases of RSV and influenza virus LRTI to determine the types of inflammatory cells present. Autopsy tissues were studied for the lymphotoxin granzyme and the apoptosis marker caspase 3. Results. Quantities of lymphocyte-derived cytokines were minimal in secretions from infants with RSV infection. Concentrations of most cytokines were greater in influenza virus, rather than RSV, infection. Lung tissues from infants with fatal RSV and influenza virus LRTI demonstrated an extensive presence of viral antigen and a near absence of CD8-positive lymphocytes and natural killer cells, with marked expression of markers of apoptosis. Conclusions. Severe infantile RSV and influenza virus LRTI is characterized by inadequate (rather than excessive) adaptive immune responses, robust viral replication, and apoptotic crisis.

Lichen planus remission is associated with a decrease of human herpes virus type 7 protein expression in plasmacytoid dendritic cells

The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.

Amino acid sequence homologies between HCV polyprotein and thyroid antigens

Recent evidence in the literature suggests that molecular mimicry between viral and self antigens may be involved in the pathogenesis of autoimmune thyroid diseases in patients with chronic hepatitis C virus (HCV) infections [1–3]. Chronic HCV infection has been reported to be associated with thyroid autoimmunity and thyroid function disorders with a mean incidence of 10% and 3%, respectively [4, 5]. Alfa-IFN therapy may exacerbate or induce underlying latent thyroid disorders, increasing the incidence of thyroid autoimmunity and thyroid function disorders to 20% and 11%, respectively [4, 5]. In keeping with the tenets of the clonal selection theory of acquired immunity, an infectious agent may circumvent the deletion of anti-self lymphocytes activating clones with receptors sufficiently degenerated to respond to mimicking epitopes and host antigens [6]. A minimum of five to six amino acids are necessary to induce an immune response, and the probability of 20 amino acids occurring in six identical residues between two proteins is 206 (for each peptide, irrespective of the sequence) or 1 in 128 000 000 [7]. We performed the comparison between the amino acid sequence of the HCV polyprotein and five tissue-specific antigens of human thyroid, available in the database on www.ncbi.nlm.nih.gov/pubmed. In particular, we examined the following HCV genotypes (with the respective NCBI sequence identification number): HCV1a (GI:130455), HCV1b (GI:130469), HCV1c (GI:385131), HCV2a (GI:130466), HCV2b (GI:130468), HCV2c (GI:555104), HCV3a (GI:514395), HCV3b (GI:676877), HCV4a (GI:402474), HCV5a (GI:2462303) and HCV6a (GI:2326455). Regarding the thyroid gland, we examined the following tissue-specific antigens: the thyroglobulin (Tg) (GI:12644093), the thyroid peroxidase (TPO) (GI:129830), the thyrotropin receptor (TSHr) (GI:136448), the sodium/iodide symporter (NaIS) (GI:12643359) and Pendrin (GI:6174895). Sequence alignments were carried out using the BLASTp, short nearly exact matches and BLASTp2 protein-protein comparison program (available at www.ncbi.nlm.nih.gov/BLAST). Amino acid sequence homologies between the HCV polyprotein and the thyroid antigens are given in detail in Fig. 1, showing the presence of identical/conservative residues in the peptides. The following proteins of the HCV polyprotein have been examined: C (capsule, core protein), E1 (envelope glycoprotein 1), E2 (envelope glycoprotein 2, NS1), p7, NS2 (non-structural protein 2), NS3 (non-structural protein 3, protease/helicase), NS4a (non-structural protein 4a), NS4b (nonstructural protein 4b), NS5a (non-structural protein 5a) and NS5b (non-structural protein 5b, RNA polymerase). The homologies between the thyroid and the viral peptides ranged from 62.5 % (five identical residues out of eight amino acids in the sequence) to 87.5% (seven identical residues out of eight amino acids in the sequence). The frequency of the homology increased up to 100%, when the conservative substitutions were included in the analysis (ten out of ten identical/conservative amino acids in the sequence, as indicated in Fig. ​Fig.11 for NaIS444 and HCV1a-NS4a1665). Fig. 1 Amino acid sequence homologies between the thyroid antigens and the HCV polyprotein (see text for abbreviations). *, Identical residues; +, conservative substitution. In each sequence, the first left-side residue inside the box corresponds to the number ... We found the presence of short peptides (eight to eleven amino acids) with a high degree of homology (62.5–100%) between the HCV polyprotein and five thyroid antigens (Tg, TPO, TSHr, NaIS and Pendrin). The homology was not restricted to a single HCV genotype or to a single thyroid antigen. The highest degree of homology was between the NaIs and the HCV1a-NS4a protein. The Tg antigen had the highest number of homologies with the different HCV genotypes. Previous studies examining 20 amino acid-length peptides showed 41.7–58.3% sequence homologies between TPO and HCV-NS5a and HCV-NS2, increased to 75.0% when including conservative/identical residues [3]. We found mimicry between the TSH-r and the N-terminal hypervariable region 1 (HVR1) of E2 in HCV1a, which is well known to be involved in chronic HCV infection [8]. The length of the short peptides is consistent with the presentation of the self/viral antigens with the I class HLA molecules to CD8 positive lymphocytes, as the II class HLA molecules usually bind longer peptides, and the mimic peptides may be involved in the acceleration of autoimmune disorders occurring in chronic HCV infection [9]. In our examination, the RLGVRATRK-HCV2b-C43 sequence presented homology with the RLGVNVTWK-Tg1361 sequence; the same viral peptide has been recently identified as a HLA-A3 supertype-restricted cytotoxic T-lymphocyte epitope in patients with HCV infections [10]. The more frequent and earlier appearance of anti-Tg antibodies in the clinical course of the thyroid autoimmunity in HCV IFN-treated patients may be related to the high number of homologies between the Tg antigen and the HCV polyprotein, whereas the anti-TPO antibodies reflect a more advanced and aggressive autoimmune thyroid destruction [11]. Further studies are necessary in order to evaluate the clinical relevance of the presence of the molecular mimicry between the HCV and the thyroid antigens in the progression of autoimmune disease.

Impaired access of lymphocytes to neoplastic prostate tissue is associated with neoangiogenesis in the tumour site

Recent reports demonstrated that neovasculature of certain murine tumours inhibits migration of lymphocytes to malignant tissues. We examined the possible existence of this phenomenon in human prostate adenocarcinoma by relating extent, patterns and composition of leucocyte infiltrates in adenocarinoma specimens (N=28) to microvessel density and percentages of these vessels expressing adhesion molecules CD54, CD106 and CD62E. Specimens of nodular hyperplasia (N=30) were used as a control for nonmalignant prostate. Increased microvessel density was detected in foci of adenocarcinoma, as compared with adjacent benign areas (P=0.004) or hyperplastic specimens (P=0.001). Only CD54 was detected on prostate vasculature; percentages of CD54-expressing vessels in adenocarcinoma lesions and adjacent areas were higher than in hyperplasia (P=0.041 and P=0.014, respectively). Infiltrating leucocytes were either scattered diffusely in tissue or organised into clusters mainly composed of CD4-positive lymphocytes; smaller percentage of tissue was occupied by clustered infiltrates in adenocarcinoma foci (mean=0.7; median=0; range=0–5) than in adjacent tissue (mean=2.5; median=1; range=0–15; P=.021) and hyperplasia (mean=1.9; median=2; range=0–5; P=.006). In adenocarcinoma foci, microvessel density tended to negatively correlate with percentage of tissue occupied by an overall leucocyte infiltrate (mean=8.6; median=7.5; range=30) and negatively correlated with percentage of tissue occupied by clustered infiltrate (P=0.045). Percentage of CD54-expressing vessels positively correlated with percentage of tissue occupied by an overall (mean=12; median=10; range=30; P=0.01) and clustered (P=0.023) infiltrate in hyperplasia, whereas in carcinoma-adjacent benign areas, correlation was detected only for clustered infiltrates (P=0.02). The results indicate that impaired access of lymphocytes to malignant lesions is associated with increased numbers of newly formed blood vessels, whereas vascular CD54 likely contributes to extravasation of lymphocytes only in benign prostate tissue.

EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges

Despite advances in surgery and adjuvant regimes, gastrointestinal malignancy remains a major cause of neoplastic mortality. Immunotherapy is an emerging and now successful treatment modality for numerous cancers that relies on the manipulation of the immune system and its effector functions to eradicate tumour cells. The discovery that the pan-epithelial homotypic cell adhesion molecule EpCAM is differentially expressed on gastrointestinal tumours has made this a viable target for immunotherapy. Clinical trials using naked anti EpCAM antibody, immunoconjugates, anti-idiotypic and dendritic cell vaccines have met variable success. The murine IgG2a Edrecolomab was shown to reduce mortality and morbidity at a level slightly lower than treatment with 5FU and Levamisole when administered to patients with advanced colorectal carcinoma in a large randomised controlled trial. Fully human and trifunctional antibodies that specifically recruit CD3-positive lymphocytes are now being tested clinically in the treatment of minimal residual disease and ascites. Although clinical trials are in their infancy, the future may bring forth an EpCAM mediated approach for the effective activation and harnessing of the immune system to destroy a pathological aberrance that has otherwise largely escaped its attention.

間質性肺炎発症から後天性免疫不全症候群と判明した2症例

Case 1: A 35-year-old man admitted for fever and respiratory failure during several weeks was found in chest computed tomography (CT) to have interstitial pneumonia, and the plasma beta-D-glucan level indicated Pneumocystis jiverocii pneumonia. Psoriasis from second-stage syphilis raised the suspicion of HIV infection. Serum anti-HIV-1 antibody proved positive and CD4-positive lymphocytes in peripheral blood were 18/microL. The man died despite treatment. Autopsy confirmed P. jiverocii pneumonia. Case 2: A 28-year-old man seen for a fever and respiratory failure was found in chest CT to have mild interstitial pneumonia. We checked for hypersensitivity pneumonitis, Mycoplasma pneumoniae pneumonia, etc. The plasma beta-D-glucan level indicated possible P. jirovecii pneumonia and immunodeficiency. Serum anti-HIV-1 antibody proved positive and CD4-positive lymphocytes in peripheral blood were 34/microL. The man was treated successfully, using trimethoprim with sulfamethoxazole for his interstitial pneumonia. His clinical symptoms were compatible with P. jirovecii pneumonia. P. jirovecii pneumonia with AIDS may present with more subacute or subtle symptoms than other immunosuppressive diseases, making it difficult to diagnose. Medical professionals should thus make it a point to familiarize themselves with AIDS prevention.

Pneumocystis Pneumonia in a Patient with Type 2 Diabetes Mellitus

A 76-year-old man, who was in the hospital for the treatment of type 2 diabetes mellitus and was receiving gonadotropin-releasing hormone (GnRH) agonist treatment for prostate cancer, developed fever and hypoxemia. Imaging revealed diffuse interstitial shadows, and PCR of the bronchoalveolar lavage fluid was positive for Pneumocystis jirovecii. The patient's absolute CD4-positive lymphocyte count dropped to 145/microl, but the HIV antibody was negative. After trimethoprim-sulfamethoxazole (TMP/SXT) treatment, the absolute CD4 positive lymphocyte count returned to normal. This patient with type 2 diabetes mellitus developed Pneumocystis pneumonia and developed a transient decrease in CD4-positive lymphocytes.

Frontal Fibrosing Alopecia in a Premenopausal Woman: Immunohistochemical and Immunofluorescence Analysis

Frontal fibrosing alopecia (FFA) was first described in 1994 and is considered a variant of lichen planopilaris in a patterned distribution that primarily affects postmenopausal women. Cicatricial alopecia of the frontoparietal hairline is characteristic; the affected area appears as a shiny, bandlike zone of incomplete hair loss with skin-coloured small hyperkeratotic papules. We describe a case of a premenopausal woman with this rare disease. To date there are very few reports of FFA in premenopausal women. The immunohistochemical investigation with lymphocytic markers (CD4, CD8, CD20, CD45RA, CD57) revealed CD8 positive lymphocytes penetrating the follicular epithelium. Direct immunofluorescence showed clumped deposition of IgM along the follicular basement membrane zone.

Lupus erythematosus: Clinical and histopathological study of oral manifestations and immunohistochemical profile of the inflammatory infiltrate

Lupus erythematosus (LE) is a multifactorial autoimmune disease, which may affect the oral mucosa in either its cutaneous and systemic forms, with varied prevalence. Forty-six patients with confirmed diagnosis of LE, presenting oral lesions were included in the study. Oral mucosal lesions were analyzed clinically, their histopathological features were investigated and inflammatory infiltrate constitution was assessed using immunohistochemistry against the following clusters of differentiation: CD3, CD4, CD8, CD20, CD68 and CD1a. From 46 patients with specific LE oral lesions 34 were females (25 with cutaneous LE and nine with systemic LE) and 12 were males (11 with cutaneous LE and one with systemic LE). Clinical aspects of lesions varied, and lips and buccal mucosa were the most affected sites. Histologically, lesions revealed lichenoid mucositis with perivascular infiltrate and thickening of basement membrane. Inflammatory infiltrate was predominantly composed by T lymphocytes of the CD4 subtype, with a minor prevalence of B lymphocytes, isolated macrophages and rare Langerhans cells. Oral lesions of lupus erythematosus show a variety of clinical aspects and histologically consist of a lichenoid mucositis with deep inflammatory infiltrate, composed predominantly of T CD4 positive lymphocytes.

Decreased CXCR4 mRNA Levels and Abnormal SDF-1 Response in Platelets from Essential Thrombocythemia.

Chemokines are soluble mediators implicated in a wide range of physiologic and pathologic processes including megakaryocytic development and platelet production. Previously, we found decreased levels of CXCR4, the only known receptor for the chemokine SDF-1, on platelet membrane from essential thrombocythemia (ET) patients, a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. In the present work we sought to evaluate the expression levels of platelet CXCR4 mRNA, the functional consequences of CXCR4 decrease in ET platelets as well as whether the receptor reduction was also extended to other lineages. We studied the expression levels of CXCR4 mRNA in platelets from ET patients by semi-quantitative RT-PCR. For semi-quantification, co-amplification of a GAPDH internal control sequence was performed. Functional studies consisted in turbidimetric evaluation of platelet aggregation induced by SDF-1 and/or ADP. In order to evaluate CXCR4 expression on leukocyte membrane, white cell subpopulations were individualized by the addition of specific MoAbs directed against CD45 labeled with PerCP and CD3 and CD14 labeled with FITC. Data were expressed as relative fluorescence intensity (RFI, ratio between the mean fluorescence intensity of CXCR4 antibody and the corresponding isotype control). The ratio between CXCR4 mRNA and the housekeeping gene GADPH in platelets from patients, 0.16 (0.1–0.24) was significantly decreased compared to controls, 0.35 (0.18–0.86), n=11, p=0.0002 (Wilcoxon signed rank sum test). Functional studies in response to SDF-1 showed a biphasic, dose-dependent full platelet aggregation in PRP in normal controls. On the contrary, abnormalities in SDF-1 response were observed in all 8 patients evaluated. Our results showed that platelet response to SDF-1 in ET patients closely paralleled that of ADP. Three patients did not respond either to SDF-1 or to ADP. From the remaining five patients, three had a delay in the second wave of aggregation induced by SDF-1 and the other two displayed only a primary wave of aggregation. These patients had a normal response to ADP when standard concentrations were used (2 mmol/L), but displayed a decreased response compared to normal controls when challenged with suboptimal doses of this agonist (0.8 to 1.2 mmol/L). CXCR4 RFI in granulocytes from patients was found elevated, 11.1 (2.22–22.74) compared to normal controls, 7.54 (1.73–9.52), p=0.036 (n=7). Similar results were found in CD3-positive lymphocytes, 40.0 (6.78–67.91) from patients and 17.23 (5.22–29.97) from normal controls, p=0.036 (Mann-Whitney Wilcoxon Rank Sum Test). On the contrary, CXCR4 on monocyte membrane was normally expressed, RFI from patients, 49.81 (1.17–118.28), and normal controls, 74.23 (3.0–80.49), p=0.8. These results indicate that the decrease in CXCR4 membrane expression on ET platelets is related to a reduced CXCR4 mRNA, and also to the abnormal platelet response to SDF-1. Besides, flow cytometric studies on leukocyte subpopulations demonstrated that CXCR4 decrease is restricted to the megakaryocytic lineage. Deregulation of SDF-1/CXCR4 axis in ET patients could be implicated in the megakaryocytic alterations seen in this illness and its presence in a megakaryocyte-related disease highlights the important role of SDF-1/CXCR4 axis in platelet development.

Photopheresis in HIV-1 Infected Patients (Pt) Using Benzoporphyrin Derivative (BPD-MA) Induces Apoptosis in CD4 Cells, Increases Cytolytic T-Cell Activity, Intracellular Expression of Chemokines, and Decreases HIV Infectivity and Viral Load.

HIV is able to elude both cellular and humoral arms of the immune system; thereby making viral control difficult. Extra corporeal photochemotherapy (ECP) or photopheresis is an immunomodulatory therapy in which lymphocytes are reinfused into the host after exposure to a photoactive compound and ultraviolet A light. It is an effective therapeutic approach to several disorders of the immune system including acute and chronic graft-versus-host disease, scleroderma, and cutaneous T-cell lymphoma. This process may offer a novel approach to viral control with minimal or no toxicity. We initiated an ex vivo and subsequently a clinical pilot trial utilizing Benzoporphyrin Derivative as the photosensitive compound. Ex vivo dosing studies identified the minimum energy levels of light exposure and concentrations of BPD that eradicated both cell-free and cell-associated HIV-1 infectivity without destroying the virus particles or infected leukocytes. Leukocytes so treated remained viable. They did demonstrate altered cytokine and chemokine expression with apoptosis induced in a minority of CD4 but not CD8 positive cells. Furthermore, there was a statistically significant increase in cytolytic T-cell activity expressed as percentage of granzyme-B release. A pilot in vivo, 24 week clinical trial in seven HIV-1 infected patients (all were required, upon entry, to have viral loads of > 10,000) using the photopheresis parameters established above demonstrated that the treatment was well tolerated and beneficial. Three individuals who had rapidly rising viral loads prior to initiating therapy stabilized once treatment began. Two of which had a (sustained) greater than .5 log decrement and 5 had stable plasma viral loads (less than a .5 log increment or decrement) with varied effects on absolute CD4 and CD8 positive lymphocytes counts. One subject achieved a greater than 1 log decrement in HIV-1 plasma viral load also developed undetectable in vivo cell-free and cell-associated HIV-1 infectivity while demonstrating an increased in vitro lymphocyte mitogen stimulation index. Subsequently, under amended and successor protocol 5 additional 12 month courses were administered to three additional patients and two of the previous enrollees. The area under the curve for viral load (viral load x # of weeks) for these twelve courses of therapy showed a significant decrease from pre to post therapy (p 0.007). There were no significant changes in CD4 or CD8 numbers area under the curve (CD4 number # of weeks and CD8 number x # of weeks). None of the subjects developed an AIDS defining illness during the course of therapy nor were there any treatment associated toxicities. These studies suggest that ECP augments activity of various arms of the immune system without any significant toxicity and may be effective in controlling HIV replication. We now plan a randomized Phase II study utilizing long-term photopheresis (twice monthly for 48 weeks) in addition to anti-retroviral therapy versus anti-retroviral therapy alone to further determine efficacy and mechanism of activity.

Ultrastructural types of alveolar macrophages in bronchoalveolar lavages from patients with pulmonary sarcoidosis

By routine applied quantitative BAL methods are particularly helpful for the diagnosis of pulmonary sarcoidosis. Here the morphology of the alveolar cells does not play a role. However, morphological and especially electron microscopic investigations might contribute to the clarification of the aetiology of this disease. In a prospective study we investigated the bronchoalveolar lavages (BALs) from 10 patients with recently histologically diagnosed, untreated pulmonary sarcoidosis. Commonly applied cytological and immunological BAL diagnostic techniques were accompanied by morphological investigations of alveolar cells, especially alveolar macrophages, using light and electron microscopy. All patients showed lymphocytic alveolitis with an increased number of CD4 positive lymphocytes as well as an increased CD4/CD8 ratio. A striking light microscopic finding was the great morphological variety of the alveolar macrophages. Electron microscopy revealed typical lymphocytes, neutrophils, and eosinophils as well as three different types of alveolar macrophages in all 10 patients: type I (approx. 30%) with a normal macrophage morphology, a vacuole-rich type II (approx. 30%) with myelin-like structures and type III (approx. 40%) with electron-dense inclusions. The occurrence of intracellular myelin figures in type II macrophages is a hint for increased phagocytotic processes of surfactant with or without its overproduction in the sense of a secondary alveolar proteinosis. Numerous electron-dense inclusions in type III also indicate an increased macrophage activity that leads to an increased release of cytokines, which in turn can trigger an inflammatory reaction.

The clinical significance of interleukin 18 assessment in sarcoidosis patients

Sarcoidosis is a multisystemic disease of unknown etiology characterized by the formation of immune granulomas in involved organs. The cytokine profile in inflamed lesions of sarcoidosis is mainly determined by T helper 1 (Th1) cells. Interleukin 18 (IL-18) is primarily a monocyte/macrophage-derived cytokine. IL-18 has been recently identified as an IFNgamma-inducing factor. The cytokine plays an important role in the induction of Th1 response and it may be responsible for sarcoidosis progression. The aim of the study was to assess the usefulness of IL-18 estimation in the sarcoidosis diagnosis and the disease course prognosis. The diagnosis of sarcoidosis was established in 88 patients (the mean age of 38.1+/-10.8 years). We measured IL-18 level in plasma and bronchoalveolar lavage fluid (BALF) cell culture supernatant (CCS) using the enzyme-linked immunoassay technique (ELISA). We also performed the flow cytometric analysis of BALF lymphocyte phenotype. Statistica 5.0 and non-parametric tests: the Mann-Whitney U-test and the Spearman correlation test, were used for statistical analysis. The patient group consisted of 55 subjects without acute symptoms of sarcoidosis, 14 patients with acute Löfgren syndrome and 19 subjects with Löfgren syndrome in the past. Lung hilar lymphadenopathy was diagnosed in 49 patients and lung interstitial changes in 39 subjects. After 6-month-observation, 49 patients were in remission, 20 subjects manifested persistent disease and 19 patients had sarcoidosis progression. Plasma IL-18 level was significantly (P<0.0001) higher in sarcoidosis patients (383+/-250pg/ml) than in control subjects (146+/-72pg/ml). Plasma IL-18 level was similar both in subjects with Löfgren syndrome and in other patients. However, IL-18 level in BALF CCS was significantly (P<0.05) lower in Löfgren syndrome patients than in subjects without acute manifestation of the disease. The highest IL-18 level in plasma was found in patients with disease progression, in subjects with lung interstitial changes and in patients with extrapulmonary manifestation of the disease. We observed a positive correlation between plasma IL-18 level and the percentage of BALF lymphocytes (R=0.202, P=0.06) as well as the percentage of activated HLA DR+T cells (R=0.23, P<0.05). There was a negative correlation between the IL-18 level in BALF CCS and the percentage of BALF CD3-positive and CD4-positive lymphocytes (R=-0.27, -0.23, P<0.05). IL-18 may play a significant role in the prolongation of sarcoidosis course. Its estimation may become a good prognostic factor, which should be analyzed together with other factors useful in sarcoidosis monitoring.

Rab33A: Characterization, Expression, and Suppression by Epigenetic Modification

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

Induction of the CXCL1 (KC) chemokine in mouse astrocytes by infection with the murine encephalomyelitis virus of Theiler

In the present study, we focused on the production of the chemokine CXCL1, also termed KC, by cultured Theiler murine encephalomyelitis virus (TMEV)-infected mouse astrocytes. cRNA from mock- and TMEV-infected cells was hybridized to the Affymetrix murine genome U74v2 DNA microarray. Hybridization data analysis demonstrated upregulation of two sequences coding for IL-8 and related to the GRO 1 oncogene MGSA. The murine counterpart of the above human genes has been reported to be the chemokine CXCL1 or KC, and therefore we studied its regulation, confirming its mRNA increase by Northern blots. The presence of CXCL1 in the supernatants of infected cells was further demonstrated by a specific ELISA and its intracellular accumulation by flow cytometry. This secreted CXCL1 was biologically active in a non species-specific way as it induces chemoattraction on human neutrophils and monocyte/macrophages, but not on CD3 positive lymphocytes. Its induction does not follow the MAP kinase pathway which transcripts are decrease in infected cells compared with uninfected astrocytes. Two inflammatory cytokines, IL-1α and TNF-α, which are also induced by TMEV in astrocytes, were potent inducers of CXCL1. Nevertheless, both mechanisms of induction follow different pathways as antibodies to both cytokines fail to inhibit TMEV-induced CXCL1 upregulation. Spinal cords but not brains from TMEV-infected SJL/J animals contain CXCL1 at the start of clinical signs of the disease. As no CXCL1 induction can be detected neither in cultured BALB/c astrocytes nor in nervous tissue, we propose an important role for CXCL1 in this experimental model of multiple sclerosis as a chemoattractant of destructive immune cells.

Virologic, hematologic, and immunologic risk factors for classic Kaposi sarcoma

Classic Kaposi sarcoma (CKS) is an inflammatory-mediated neoplasm that develops in the presence of KS-associated herpesvirus (KSHV) and immune perturbation. In the current study, the authors compared CKS cases with age-matched and sex-matched KSHV-seropositive controls without human immunodeficiency virus-1 infection and markers of viral control, blood counts, CD4-positive and CD8-positive lymphocytes, and serum beta-2-microglobulin and neopterin levels. Viral loads were detected using real-time amplification of the KSHV-K6 and EBV-pol genes, anti-K8.1 (lytic) titers were detected by enzyme-linked immunoadsorbent assay, and antilatent nuclear antigen (LANA) titers were detected using immunofluorescence. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using logistic regression adjusted for sex, age, and study site. Peripheral blood mononuclear cells (PBMC) KSHV DNA detection (P < or = .0001) and high KSHV lytic (>1:1745; P < or = .0001) and latent (>1:102,400; P = .03) antibody titers were found to be positively associated with CKS risk. Antibody titers were higher in cases with lesions compared with cases without lesions (P < or =.05). The detection of Epstein-Barr virus (EBV) DNA in PBMCs was not found to be associated with CKS (P = .95). Independent of PBMC KSHV DNA, CKS risk was found to be positively associated with reduced hematocrit (<37.4%; P = .03), hemoglobin (<12g/dL; P = .04), and lymphocytes (<1000 cells/microL; P = .004), including CD4-positive (+) cells (<457 cells/microL; P = .07) and CD8+ cells (<213cells/microL; P = .04), and with increased monocytes (> or =638 cells/microL; P = .009). Nonsignificant elevations of beta-2-microglobulin and neopterin were observed among cases regardless of disease burden (P > or = .08). In a multivariate model, the CKS risk was found to be associated with PBMC KSHV DNA (OR of 2.7; 95% CI, 1.4-5.3), a high KSHV lytic antibody titer (OR of 3.7; 95% CI, 1.9-7.4), and low lymphocytes, particularly among those patients age <70 years (OR of 8.0; 95% CI, 2.7-23.7). The findings of the current study appear to corroborate the specificity of KSHV and highlight the hematologic and immunologic correlates involved in the pathogenesis of CKS.

Immunosuppression in patients with severe acute pancreatitis

In severe acute pancreatitis (SAP), immunologic impairment in the early phase may be linked to subsequent infectious complications. In this study, immunologic alterations in patients with SAP were analyzed, and immunologic parameters related to infectious complications were clarified. A total of 101 patients with SAP were analyzed retrospectively. Various immunologic parameters on admission were analyzed and compared between the infection group and noninfection group during SAP. Furthermore, chronologic change in the lymphocyte count was investigated, and its utility for predicting infection was compared with conventional scoring systems. Serum immunoglobulin G (IgG), serum IgM, lymphokine-activated killer cell activity, and natural killer cell activity were low, and the incidence of abnormally low values was 50.0%, 65.0%, 45.5%, and 42.4%, respectively. Serum complement factor 3 was significantly negatively correlated with the APACHE II score. The lymphocyte count was decreased below the normal range, and was significantly negatively correlated with the APACHE II score. CD4-, CD8-, and CD20-positive lymphocyte counts were below the normal range, and CD4- and CD8-positive lymphocyte counts were significantly lower in the infection group. The lymphocyte count on day 14 after admission was significantly lower in the infection group and was more useful for predicting infection than conventional scoring systems. Immunosuppression occurs from the early phase in SAP, and quantitative impairment of lymphocytes, mainly T lymphocytes, may be closely related to infectious complications during SAP. CD4- and CD8-positive lymphocyte counts on admission and the lymphocyte count on day 14 after admission may be useful for predicting infection.

Reduced CD1d Expression in Colonic Epithelium in Microscopic Colitis

CD1d is a member of a major histocompatibility complex class I-like molecule family. Its function may involve presenting microbial nonpeptide or lipidic antigens to T lymphocytes, therefore to serve as an important factor in normal mucosal immunity of the gastrointestinal tract. In this study, the expression level of CD1d in microscopic colitis (ie, collagenous and lymphocytic colitis) was examined, and compared with that in normal colonic mucosa. Formalin-fixed, paraffin-embedded colon biopsies with diagnosis of lymphocytic colitis (19 cases), collagenous colitis (6 cases), and no pathologic change (20 cases) were studied immunohistochemically using monoclonal antibodies against human CD1d, CD3, CD4, and CD8. CD1d staining in the epithelium and lamina propria was graded along a scale of 0 to 4. Intraepithelial CD3-positive lymphocytes were counted in an area of 300 epithelial cells for each specimen. The results show that CD1d was expressed in normal colonic epithelial cells, primarily on the basolateral membranes with a concentrated intracellular pool in the subnuclear region. The expression level was markedly reduced in both lymphocytic colitis (P<0.001) and collagenous colitis (P<0.001), along with a significant increase in the number of intraepithelial CD3/CD8 lymphocytes (P<0.001). These findings suggest that microscopic colitis is associated with decreased epithelial expression of CD1d, an important immunoregulatory molecule in the gastrointestinal tract.

Single platform enumeration of lymphocyte subsets in healthy Indians aged between 18 and 49 years

Single platform enumeration of lymphocyte subsets in healthy Indians aged between 18 and 49 years