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Invasive cervical cancer is caused by human papillomavirus (HPV).To describe the prevalence and genotype distribution of HPV types in women at risk for cervical neoplasia.Our study summarized HPV types detected in 6654 samples that were sent to the serology laboratory from cervical clinics in northern Israel between 2006-2014. The HPV test was performed during investigation of atypical squamous cells of undetermined significance (ASCUS) results on Pap tests or due to complaints suggestive of cervical neoplasia. HPV types were classified as high risk (HPV-HR) and low risk (HPV-LR).Of the samples, 46.4% (3085/6654) were HPV-HR positive. Of women with cervical intraepithelial neoplasia 2-3 (CIN 2-3) or cancer, 292/318 (91.8%) and 137/145 (94.5%), respectively, were HPV-HR positive. HPV 16 and HPV 18 were detected in 11.8% of the total samples and in 48.2% and 64.9% of the women with CIN 2-3 and with cancer, respectively. HPV was negative in 8/145 (5.5%) and 26/318 (8.2%) of women with cervical cancer and CIN 2-3, respectively.This study shows the prevalence of HPV types in women at risk for cervical neoplasia. The sensitivity of all HPV types for CIN 2-3 and cervical cancer was 91.8% and 94.5%, respectively; and of HPV-HR types, 89% and 92.4%, respectively. Triage of HPV-HR types should be considered in women with ASCUS because HPV-HR types were discovered in only 36.7%. The distribution of HPV types in our population is similar to that reported for other developed countries.

We evaluated the effects of hyperoxia on pulmonary inflammatory changes in sepsis induced by cecal ligation and puncture (CLP) in rats. Seven groups were studied: sham-operated rats breathing air for 20 or 48 h; CLP breathing air for 20 or 48 h; and CLP + 100% oxygen for 20 h, or 70% oxygen for 48 h, or 100% oxygen intermittently (6 h/d) for 48 h. Video microscopy was used to monitor lung macromolecular leak, microvascular flow velocity, and shear rates, and lung morphometry was used for leukocyte infiltration and solid tissue area. Cell counts, tumor necrosis factor α, and nitrites were determined in peripheral blood and lung lavage fluid. Expression of adhesion molecules in blood leukocytes was evaluated by flow cytometry. Cecal ligation and puncture induced inflammation manifested in leukopenia, left shift, thrombocytopenia, increased expression of L selectin and CD11, increased serum and lavage fluid tumor necrosis factor α and leukocytes, and increased lung tissue area, macromolecular leak, and sequestration of leukocytes. Inhalation of 100% oxygen for 20 h increased nitrites (P < 0.01) and decreased leukocyte count in lavage fluid (P < 0.05) and attenuated lung macromolecular leak and changes in solid tissue area (P < 0.01). Inhalation of 70% oxygen (48 h) attenuated expression of adhesion molecules (P < 0.001) but failed to attenuate markers of lung inflammation. In contrast, intermittent 100% oxygen exerted favorable effects on markers of inflammation, attenuated leukocyte expression of L selectin and CD11 (P < 0.01), decreased pulmonary sequestration of leukocytes (P < 0.001), and ameliorated changes in macromolecular leak (P < 0.01) and lung solid tissue area (P < 0.05). Our data support the beneficial effects of safe subtoxic regimens of normobaric hyperoxia on the systemic and pulmonary inflammatory response following CLP.

Transplantation involves preoperative ischemic periods that contribute to endothelial cell (EC) dysfunction and T-cell activation, leading to graft rejection. As hypoxia is a major constituent of ischemia, we evaluated its effect on the ability of ECs to express HLA-DR, which is required for presentation of antigens to T cells, and by itself serves as an important target for allogeneic T cells. Primary human umbilical vein ECs (HUVEC) and the human endothelial cell line EaHy926 were incubated in normoxia or hypoxia (PO(2) < 0.3%). Hypoxia increased the membranal expression (by 4-6 fold, P < 0.01) and secretion (by sixfold, P < 0.05) of HLA-DR protein, without influencing the accumulation of its mRNA. Alternative splicing, attenuated trafficking, or shedding from the plasma membrane were not observed, but the lysosomal inhibitor bafilomycin A1 reduced HLA-DR secretion. Hypoxia-induced endothelial HLA-DR elevated and diminished the secretion of IL-2 and IL-10, respectively, from co-cultured allogeneic CD4(+) T cells in a HLA-DR-dependent manner, as demonstrated by the use of monoclonal anti-HLA-DR. Our results indicate a yet not fully understood post-translational mechanism(s), which elevate both membranal and soluble HLA-DR expression. This elevation is involved in allogeneic T-cell activation, highlighting the pivotal role of ECs in ischemia/hypoxia-associated injury and graft rejection.

Glaucoma filtering surgery may be compromised by cystic blebs which develop more frequently when anti-metabolites are used to arrest wound healing. Matrix metalloproteinases (MMPs) and the naturally occurring tissue inhibitors of metalloproteinases (TIMPs) are essential in connective tissue remodeling and wound healing. This study aimed to determine whether filtering blebs display increased expression of MMP-2, MMP-9, TIMP-1 and TIMP-2, and whether it is reflected in tear fluid. Tissue samples from leaking blebs (n = 5) and control conjunctiva (n = 5) were evaluated by immunohistochemistry for MMP-2, MMP-9, TIMP-1 and TIMP-2. Tear fluid was collected from 12 patients (12 eyes) with cystic blebs and ten patients (ten eyes) with flat blebs following trabeculectomy with Mitomycin C applied and 16 controls. MMP levels were evaluated by zymography and TIMP levels by Western blot analysis. Conjunctival tissue was obtained from five eyes with cystic leaking blebs and five control eyes undergoing cataract surgery. More extensive MMP-2 and MMP-9 expression was found in the epithelial and stromal layers of blebs than in control conjunctiva. TIMP-1and TIMP-2 were expressed in all layers of the blebs, but only in the epithelium of control conjunctiva. MMP-2 and proMMP-2 activity in tears from eyes with flat blebs was significantly higher than that of controls, while activity in tears of eyes with cystic blebs was significantly higher than in those with flat blebs. There was no difference in MMP-9 activity between tears of control and post-filtering surgery eyes. Increased MMPs and TIMPs expression is associated with the formation of filtering blebs, suggesting involvement of MMPs in bleb remodeling. MMP-2 and ProMMP-2 levels in tear fluid may be markers for bleb configuration.

Hypoxia, which characterizes ischemia, trauma, inflammation, and solid tumors, recruits monocytes, immobilizes them, and alters their function, leading to an anti-inflammatory and proangiogenic phenotype. Monocyte extravasation from the circulation and their migration in tissues are partially mediated by the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The mechanisms evoked by hypoxia that regulate monocyte migration and activation are not entirely clear. Specifically, the effect of hypoxia on TIMPs in these cells has hardly been investigated. We show that hypoxia reduces TIMP-2 secretion from human primary monocytes and from the monocyte-like cell lines U937 and THP-1 by three- to fourfold ( P < 0.01), by inhibiting TIMP-2 transcription through mechanisms that involve the transcription factor SP-1. Hypoxia also lowers TIMP-2 protein secretion from human endothelial cells (by 2-fold, P < 0.05). TIMP-2 levels do not influence the reduced migration of THP-1 cells in hypoxia; however, low TIMP-2 levels enhance endothelial cell migration/proliferation, their ability to form tubelike structures in vitro, and the appearance of mature blood vessels in a Matrigel plug assay in vivo. Thus we conclude that reduced TIMP-2 levels secreted from both hypoxic monocytes and endothelial cells are proangiogenic.

Detachment of endothelial cells (ECs) from the extracellular matrix (ECM) is required not only for angiogenesis, but also for EC apoptosis. Matrix metalloproteinase (MMP)-2 plays a major role in the degradation of the ECM, supporting an essential role for this enzyme in both survival (angiogenesis) and death of ECs. Our aim was to study these seemingly paradoxical effects of MMP-2. We rationalized that inhibiting apoptosis would drive MMP-2 toward a prosurvival activity, clarifying the mechanisms involved. By employing specific inhibitors to two major apoptotic pathways in ECs, caspases and p38 MAPK (p38), we demonstrated that they differently affected EC behavior as well as MMP-2 expression. The p38 pathway appears to enhance MMP-2 synthesis, its partial (“intermediate”) and its full activation, probably via membrane type (MT)1-MMP, while caspases enhance MMP-2 synthesis and full activation but reduce MT1-MMP and MMP-2 intermediate form. Evaluation of the reciprocal influences of MMP-2 on ECs showed that the intermediate form supported survival and migration, and the fully active form led to cell death. In addition, a pro- and intermediate form-rich environment, even in the presence of the fully active form, exerted protective effects. Thus the seemingly conflicting effects of MMP-2 on EC survival may be explained by the ratio between the MMP-2 activation forms. A regulatory loop between active MMP-2 and p38 but not between MMP-2 and caspases was also observed, suggesting that MMP-2 is downstream to caspases where it serves as an “exterminator” molecule. Altogether, modification of caspase and p38 pathways, via changes of local MMP-2, affect survival and angiogenic steps in ECs.

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Objective: Inhalation of oxygen improves the hemodynamic status and attenuates the inflammatory response after intestinal ischemia-reperfusion (IR). Yet, the use of hyperoxia is hindered by concerns that it could exacerbate reperfusion injury by increasing free radical formation. We examined the effect of hyperoxia on enterocyte turnover and intestinal preservation and rehabilitation following IR injury in rats. Design: Animal study. Setting: Research laboratory. Subjects: Male Sprague-Dawley rats. Interventions: Animals were assigned to four experimental groups: 1) Sham rats underwent laparotomy without vascular occlusion and breathed air, 2) Sham-oxygen rats underwent laparotomy without vascular occlusion and breathed 100% oxygen, 3) IR rats underwent occlusion of the superior mesenteric artery and portal vein for 30 minutes and breathed air, and 4) IR group treated with oxygen (IR-O 2 )rats underwent IR and breathed 100% oxygen starting 10 minutes before and continued for the first 6 hours after reperfusion. Intestinal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 hours after IR. Measurements and Main Results: In IR rats, breathing 100% oxygen resulted in a significant decrease in Park's injury score in the ileum (p < 0.05 from untreated IR rats). Rats treated with oxygen also demonstrated a significant increase in mucosal weight(p < 0.05) and mucosal DNA (p < 0.05) in the jejunum and ileum, and an increase in villus height (p < 0.01), and crypt depth (p < 0.05) in the ileum. Enterocyte proliferation (assessed by bromodeoxyuridine uptake) was significantly decreased in the jejunum and ileum in untreated IR rats. Oxygen therapy increased enterocyte proliferation in the ileum, and diminished both the apoptosis index and Bax gene expression in the jejunum and ileum (p < 0.05). Plasma thermochemiluminescence oxidizability assay revealed significantly higher thermochemiluminescence ratios in IR group treated with oxygen than in untreated IR rats (p < 0.05) at 6 hours postreperfusion suggesting a significantly lower prior in vivo molecular oxidation. Conclusions: Hyperoxia reduces small bowel injury, accelerates enterocyte turnover, and improves intestinal rehabilitation after IR.

Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-α (TNF-α) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O2< 0.3%) reduces the secretion of LPS-induced TNF-α ( P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-α mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-α by twofold ( P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-α by twofold ( P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-α in hypoxic peritoneal macrophages.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) mediates tumor cell-macrophage interactions, and has been shown to induce both matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). However, the epitope responsible for MMP induction is controversial, and the epitope responsible for VEGF induction is yet unknown. We generated a novel anti-EMMPRIN antibody directed against a specific epitope that successfully inhibited the production of both MMP-9 and VEGF in tumor cell-macrophage in vitro co-culture systems, exhibiting a U-shaped dose response. Furthermore, this antibody efficiently inhibited in vivo tumor progression in both the RENCA renal cell carcinoma and CT26 colon carcinoma subcutaneous tumor models, and reduced tumor size and number of metastatic foci in the 4T1 orthotopic model. This was achieved by inhibiting angiogenesis as assessed by immunohistochemical staining for the endothelial marker CD31, by inhibiting tumor cell proliferation as assessed by the staining for Ki-67, and by enhancing tumor cell apoptosis as assessed in the TUNEL assay. Moreover, administration of the antibody recruited more macrophages into the tumor, and skewed the tumor microenvironment for macrophages from TGFβ-dominated anti-inflammatory microenvironment, to a less immunosuppressive one. The antibody improved the ability of stimulated macrophages to perform antibody-dependent cell cytotoxicity (ADCC) and kill tumor cells. Thus, our new antibody maps the epitope capable of inducing both MMPs and VEGF, and places EMMPRIN as a good target for cancer therapy.

The involvement of matrix metalloproteinases (MMPs) in ischemic tissue damage and remodeling has been reported by many investigators. Our study was designed to investigate the involvement of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in rat retinal ischemic injury, the effect of nitric oxide synthase (NOS) inhibitors on MMPs’ activity in this model and whether minocycline (an MMP inhibitor) is protective in retinal ischemia. Ninety-four rats were used in the study. Ischemia was induced by 90 min elevation of intraocular pressure. MMPs’ activities and the effect of NOS inhibitors [aminoguanidine (AG) or N-nitro-L-arginine (NNA)] and minocycline on MMPs’ activities were assessed by zymography and TIMPs expression by Western analysis. Morphological damage was quantified by morphometry of hematoxylin and eosin-stained retinal sections. Retinal extracts exhibited activities of proMMP-9 and proMMP-2. The activity of proMMP-9 increased immediately post ischemia (PI) and peaked to 4.6 times that of normal untreated controls in ischemic retinas and to 2.6 times that of controls in retinas of fellow sham-treated eyes at 24 h PI. The relative amount of TIMP-1 increased to 1.9-fold following ischemia and 2.5-fold in fellow sham-treated eyes at 24 h PI. ProMMP-2 activity increased more than two-fold immediately, at 24 h and at 48 h PI in ischemic retinas, and insignificantly in fellow sham-treated eyes. Treatment with 25 mg/kg AG or NNA caused a non-significant increase in proMMP-9 activity at 24 h PI (3.7- and 2.9-fold, respectively, p>0.6). There was no effect of AG or NNA on the activity of proMMP-2. Minocycline significantly attenuated the retinal ischemic damage, primarily by partially preserving ganglion cells and the inner plexiform layer. Minocyline (0.5 mg/ml or 5 mg/ml) inhibited MMPs’ activities in ischemic retinal extracts in vitro. MMPs participated in morphological ischemic damage to rat retina. Treatment with minocycline dramatically attenuated damage to the retina.

In winter 2001, an outbreak of pertussis involving an estimated 75 people occurred among soldiers serving in an infantry regiment of the Israeli Defense Forces (IDF). Nasopharyngeal swabs were obtained from patients and contacts for culture and PCR. Serum samples were obtained and assayed by ELISA for the presence of IgA, IgM and IgG antibodies to a lysate antigen of Bordetella pertussis. The calculated attack rate was 21% based on clinical signs alone (cough lasting 30 days or longer) and 9·5% based on clinical signs with laboratory confirmation (by PCR, IgA or IgM). A high carriage rate was observed; 20% of the asymptomatic and previously symptomatic subjects were PCR-positive for B. pertussis. These findings emphasize the importance of B. pertussis as a causative agent of epidemic respiratory infections in young adults and reveal the occurrence of a significant proportion of pertussis transient carriers during an outbreak of the disease.

Reciprocal interactions between T cells and antigen-presenting cells (APCs) within the ‘Immunological-Synapse’ (IS) govern immune cell autoreactivity in multiple sclerosis (MS). The present study examined the expression of a range of co-stimulatory molecules: CD40, CD54, CD80, CD86 and HLA-DR, on the cell-surface of CD14+ peripheral blood monocytes (PBM) from relapsing–remitting (RR) and secondary-progressive (SP)-MS patients, prior to and during 1 year of Interferon (IFN)-β-1a (Rebif®) therapy. Prior to treatment, patients from both MS subtypes expressed elevated CD80 and reduced CD40 levels in comparison to controls. CD86 expression was significantly reduced in SP compared to RR patients and controls. IFN-β therapy led to a significant reduction in the expression of CD54 and CD80 in both groups of patients as well as to elevation of CD40 and CD86 expression in SP patients. These results confirm IFN-mediated modulation of the APC surface within the immunological-synapse and implicate CD80 and CD86 as targets for interventional therapies in MS as well as other Th1-mediated autoimmune diseases.

Monocytes/macrophages in ischemic tissues are involved in inflammation and suppression of adaptive immunity via secretion of proinflammatory cytokines and reduced ability to trigger T cells, respectively. We subjected human mononuclear cells and mouse macrophages to hypoxia and reoxygenation, the main constituents of ischemia and reperfusion, and added lipopolysaccharide (LPS) to simulate bacterial translocation, which frequently accompanies ischemia. We monitored the secretion of tumor necrosis factor α (TNF-α) and the surface expression of human leukocyte antigen-DR and the costimulatory molecules CD80 and CD86 on monocytes/macrophages. Hypoxia selectively reduced the surface expression of CD80 (P

High prevalence of coeliac disease (CD) has been reported in various autoimmune disorders, buthas not been studied in the antiphospholipid syndrome (APS). We aimed to establish the prevalence of CD antibodies in a cohort of APS patients, and to examine whether CD may be responsible for some of the manifestations of APS. Fifty-seven patients (47 females, 10 males) with APS were studied for clinical manifestations and serological markers of the disease, as well as the presence of anti-endomysial antibodies using an ELISA assay (EMA-ELISA). Control subjects were 171 healthy individuals, age- and sex-matched (141 females). Eight patients with APS (14%, six females) were found to have EMA-ELISA antibodies, compared with 2/141 (1.1%) of controls (P = 0.0003). Antibodies against beta2-glycoprotein-I (beta2GPI) epitopes (GRTCPKPDDLP) were more prevalent in EMA-positive patients than in EMA-negative patients (P = 0.006). Vasculitic skin lesions were significantly more common in EMA-ELISA-positive compared with EMA-ELISA-negative patients(62.5 versus 16.3%, P = 0.01). Among the skin manifestations, superficial cutaneous necrosis (37.5 versus 2%, P = 0.007) was more prevalent in EMA-ELISA-positive than in EMA-ELISA-negative patients. EMA-ELISA antibodies are common in APS, and their presence is associated with high prevalence of antibodies recognizing certain beta2-glycoprotein epitopes, and with cutaneous manifestations of APS.

The combined expression of the inflammatory mediators, matrix metalloproteinases (MMPs), soluble form of intracellular adhesion molecule ICAM-1 (sICAM-1) and interleukin (IL)-8, was evaluated in children infected with bacterial or viral meningitis. MMP-2 and IL-8 were detected in all CSF samples and were enhanced in both bacterial and viral infected samples, compared to those from control children. The expression of MMP-9 as well as sICAM-1 was not detected in control CSF while observed in viral infected and further elevated in bacterial infected samples. This pilot study supports a role for MMPs, IL-8 and sICAM in infectious meningitis and suggests further research to determine their possible use as biomarkers for various forms of meningeal infection as well as the use of their specific antagonists as potential therapeutic agents for central nervous system (CNS) inflammatory processes.

Objective This study examined the involvement of different matrix metalloproteinases (MMPs) in articular cartilage in the process of growth, maturation, and aging of mice, and compared the temporal changes in the expression of MMPs between temporomandibular joints (TMJ) and knee joints. Methods Homogenates of intact tibial plateau, femoral condyle, and TMJ condyle cartilages from animals of different ages were assessed for gelatinase (MMP-2 and MMP-9) activity by zymography. The messenger RNA (mRNA) expression of MMPs 1, 2, 3, 9, and 13 in tibial plateau cartilage was determined by semiquantitative reverse transcription–polymerase chain reaction, and immunohistochemistry was used to localize MMPs 2, 3, 9, and 13 in the knee joints and TMJ from mice of different ages. Results The pattern of gelatinase (MMP-2 and MMP-9) activity and their protein expression as well as that of MMPs 3 and 13 varied with the age of the mouse, and differences in expression were observed between the knee and TMJ cartilage. The expression of mRNA for the MMPs in the tibial plateau was also age related. Conclusion This study demonstrated changes in the protein and mRNA expression of MMPs 2, 9, 3, and 13 during growth, maturation, and aging in mice. The temporal changes were characteristic of the joint, and distinct differences were observed between the TMJ and knee cartilage. The differences in temporospatial expression of MMPs between the knee joint and TMJ may be the result of differences in load and function of these joints. The information provided in this study contributes to a better understanding of the role of these MMPs in the maintenance and integrity of cartilage tissue.

The matrix metalloproteinases (MMP) are enzymes crucial for the physiological patrol as well as pathological chemotaxis of immune cells to target tissues. The present study examined differential effects of pro-inflammatory [IL-18, IL-12 and tumor necrosis factor (TNF)-alpha] versus anti-inflammatory (IL-4) cytokines on the modulation of MMP and their endogenous tissue inhibitors (TIMP) expression in the U937 cell line. IL-18 and IL-12 separately and synergistically enhanced MMP-2, while TNF-alpha led to the elevation of MMP-9. All pro-inflammatory cytokines enhanced MT1-MMP expression and IL-4 suppressed TNF-alpha-induced MMP-9 expression. This study demonstrated that elevated IL-18 and IL-12, and related pro-inflammatory activity, may be associated with aberrant MMP activity, suggesting modulation of MMP expression using IL-12 and IL-18 antagonists as future therapeutic strategies to attenuate inflammatory and autoimmune disorders.

Inorganic lead (Pb) is a ubiquitous environmental contaminant that produces a variety of deleterious effects in the central nervous system (CNS). Matrix metalloproteinases (MMPs), specifically MMP-9, induced by inflammatory cytokines, are increasingly being implicated in CNS pathology. The present study demonstrates that low concentrations of either pro-inflammatory cytokines (TNF-α and IL-1β) or Pb did not influence the MMP-9 expression in a glial cell line (C6) when added separately. However, combined administration of Pb and cytokines induced a marked synergized elevation of MMP-9 expression in spite of a reduction in the number of glial cells. These results demonstrate a possible new mechanism by which Pb may induce neuropathological processes.

PROBLEM: Our objective in this study was to correlate immunosuppressive properties of follicular fluid (FF) and media conditioned by zygotes and early embryos with the occurrence of conception in an in vitro fertilization (IVF) program. METHOD OF STUDY: Fifty-seven IVF patients were studied. Donor lymphocytes were incubated with mitogens and FF from mature oocytes or conditioned media from zygotes and early embryos. Proliferation was assessed by radioactive thymidine incorporation. Proliferation Index (PI) was the ratio between radioactive labeling of lymphocytes incubated in the presence and absence of a mitogen. RESULTS: The FFs and media conditioned by zygotes from conception cycles had higher immunosuppressive activity than those from non-conception cycles. CONCLUSIONS: Immunosuppressive activity present in FF and media conditioned by zygotes may be a major determinant of conception in IVF, and may serve as a marker for embryo quality.

BACKGROUND Degradation of the extracellular matrix (ECM) is essential for progression and metastasis of cancer cells. The ECM-degrading enzymes, matrix metalloproteinases (MMPs), are produced mainly by intratumor monocytes/macrophages. MMPs, particularly MMP-9, are reported to be of crucial significance for both growth and tumor invasiveness. Inhibition of the expression of MMP-9 may prevent tumor development. High-dose intravenous gamma globulins (IVIG) effectively inhibit metastatic spread of tumors in mice and humans and a variety of mechanisms have been suggested to explain this effect. METHODS We studied the effect of purified IVIG on MMP-9 secretion and mRNA expression by in vitro differentiated human monocytic cells (cell lines and peripheral blood monocytes). Zymography was employed to measure gelatinase secretion and Northern blot analysis was used to detect mRNA expression. Involvement of F(ab)2 and Fc components in IVIG activity was also evaluated. RESULTS IVIG dose dependently and significantly reduced the amount of secreted MMP-9 and its mRNA expression. F(ab)2, but not Fc fragments, led to suppressed MMP-9 activity. However, competitive experiments demonstrated that Fc, but not F(ab)2 fragments, reversed the IVIG-induced inhibitory effects. CONCLUSIONS These results suggest that the whole IgG molecule may be needed for pertinent IVIG-induced MMP-9 down-regulation. This study points to an additional new mechanism whereby IVIG may play a beneficial role in the prevention of tumor spread in humans. Cancer 2002;95:2032–7. © 2002 American Cancer Society. DOI 10.1002/cncr.10905

OBJECTIVES: Recent studies suggest that celiac disease was previously underdiagnosed. To find out whether antiendomysial antibodies underestimate the prevalence of celiac disease, we elected to use a strategy combining multiple serological markers to explore the prevalence of celiac disease in Israel and the usefulness of the various antibodies in screening for celiac disease. METHODS: Serum samples from 1571 healthy blood donors were tested. A small intestinal biopsy was offered to all patients who tested positive for either human tissue transglutaminase antibodies, an ELISA kit based on antiendomysium (EMA-ELISA), immunoglobulin A antigliadin verified by antiendomysial immunofluorescence antibodies, and to patients who were IgA deficient with elevated antigliadin IgG. RESULTS: A total of 59 subjects (3.8% of study population) were offered an intestinal biopsy based on serological findings, and 30 of 59 patients agreed to undergo intestinal biopsy (1.9% of study population). Celiac disease was diagnosed in 10 patients, establishing a prevalence of at least 1:157 in the general population (0.6%, CI = 0.3–1.1%). Using any serological marker alone would have underestimated the prevalence of celiac disease, as it was diagnosed in only two patients who tested positive for endomysial immunofluorescence antibodies (prevalence of 1:786, 0.1%, CI = 0.02–0.5%), six patients positive for tissue transglutaminase (prevalence of 1:262, 0.4%, CI = 0.1–0.9%), and seven patients positive for ELISA-EMA (prevalence of 1:224, 0.45%, CI = 0.2–0.9%). CONCLUSIONS: The prevalence of celiac disease in Israel is at least 1:157 in the general population, confirming its underdiagnosis in previous studies. The disparity between the various serological markers suggest that the use of one serological marker is insufficient for establishing the true prevalence of celiac disease.

Among the consequences resulting from the exposure of endothelial cells (ECs) to ischemia/reperfusion is angiogenesis, involving degradation of vascular basement membrane and extracellular matrix. Matrix metalloproteinase (MMP)-2, a member of the MMP family, partakes in this process. MMP-2, secreted as a proenzyme, undergoes activation through interaction with membrane type (MT)1-MMP and the endogenous tissue inhibitor of MMPs (TIMP)-2. Although hypoxia and reoxygenation (H/R) are major constituents of ischemia/reperfusion processes, their direct effects on endothelial MMP-2 have been scarcely investigated. This study examined the in vitro effects of H/R on human macrovascular ECs (EAhy 926). The level of MMP-2 mRNA (Northern blot) and protein (zymography, ELISA) and the mRNA of its activator (MT1-MMP) and inhibitor (TIMP-2) were analyzed. Short (6-hour) hypoxia inhibited the mRNA expression of MMP-2, MT1-MMP, and TIMP-2, culminating in reduced latent and active MMP-2 protein. Prolonged (24-hour) hypoxia further suppressed MT1-MMP and TIMP-2 mRNA, whereas it enhanced MMP-2 mRNA and enzyme secretion (after 48-hour hypoxia). Reoxygenation did not influence the inhibited TIMP-2 but upregulated MMP-2 and MT1-MMP mRNA expression, leading to enhanced secretion of active MMP-2 protein. These results demonstrate H/R-mediated modulation of EC MMP-2 at both transcriptional and posttranscriptional levels. Prolonged hypoxia of ECs appears to enhance MMP-2 production and secretion, whereas reoxygenation further increases its level. These H/R-mediated effects on MMPs have the potential of enabling EC migration and possible angiogenesis.

Expression of MHC class II molecules is restricted to professional antigen-presenting immune cells, but it can be induced by IFN-gamma in other cell types. Thyroid cells have been shown to induce class II expression (mainly HLA-DR) following stimulation with IFN-gamma and addition of tumor necrosis factor (TNF)-alpha synergistically enhanced this expression. Class II transactivator (CIITA) has been implicated as the master regulator of MHC class II molecules and its transcription has been shown to be regulated from four different promoters, one of which is responsible for its induction by IFN-gamma. The aim of this study was to find whether CIITA is synergistically induced by IFN-gamma and TNF-alpha in the human thyroid MRO-87-1 cell line, and to investigate the molecular mechanisms responsible for this synergism. We have demonstrated that IFN-gamma and TNF-alpha synergistically induce HLA-DRalpha and CIITA mRNAs, but prolonged incubation resulted in the inhibition of CIITA mRNA accumulation. Several potential mechanisms that could explain the synergistic effect were explored. NF-kappaB did not bind the CIITA inducible promoter and addition of SN50, which inhibits NF-kappaB translocation to the nucleus, did not change the synergistic effect. Furthermore, IFN-gamma did not induce IkappaBalpha degradation. Synergistic activation of signal transducer and activator of transcription (STAT)-1 or IFN regulating factor (IRF)-1 was not observed, and STAT-1 did not bind the CIITA inducible promoter. IRF-1, although not synergistically induced or activated, bound synergistically to its specific cis element on the CIITA type IV promoter. Thus we propose that IRF-1 binding mediates the synergistic induction of HLA-DRalpha and CIITA in thyroid cells.

Telomerase activity compensates for the erosion of chromosomes and it has been detected in a wide variety of human tumors. Cytokeratin 20, an intermediate filament of epithelial cells, is expressed particularly in the urinary tract. These 2 molecules are candidates to become markers for the detection and followup of bladder carcinoma. We evaluate whether each molecule may serve as a potential marker and whether the 2 combined would improve the detection or followup of bladder carcinoma in a noninvasive manner.We obtained 44 morning urine samples from patients with transitional cell carcinoma patients and 26 from age matched patients with a wide variety of clinical disorders but no malignancy of any kind. A telomerase polymerase chain reaction-enzyme-linked immunosorbent assay kit was used to determine telomerase activity and cytokeratin 20 expression was determined by nested reverse transcriptase-polymerase chain reaction.All samples tested positive for cytokeratin 8 expression, which verified epithelial cells in the urine samples. Of the 44 transitional cell carcinoma cases of all stages and grades 37 (84.1%) were positive for telomerase activity, 36 (81.8%) were positive for cytokeratin 20 expression and 65.9% were double positive. Of the 29 controls with various clinical conditions other that malignancy 22 (75.9%) were positive for telomerase activity, 13 (44.83%) were positive for cytokeratin 20 expression and 34.6% were double positive.Telomerase activity and cytokeratin 20 expression are not specific for malignancy and may be detected in many nonmalignant pathological conditions. Therefore, their use as potential markers of bladder carcinoma should be carefully reevaluated.

Matrix metalloproteinases (MMPs) have recently been implicated in the pathogenesis of multiple sclerosis. Their suggested role includes the disruption of the blood-brain barrier, immune cell transmigration into the central nervous system, and myelin degradation. The present study characterized the mRNA level of a wide spectrum of MMPs and tissue inhibitors of metalloproteinases (TIMPs) expressed by peripheral blood leukocytes from relapsing-remitting (n = 16) and secondary-progressive (n = 12) multiple sclerosis patients. The expression of the same MMPs and TIMPs was evaluated also in a prospective 12-month follow-up of 6 patients randomly chosen from each of the 2 groups during interferon beta-1a treatment. Reverse transcription-polymerase chain reaction assessment demonstrated elevated levels of MT1-MMP and MMP-7 mRNA levels in both groups of patients, and no significant differences in MMP-9 levels, compared with healthy controls. Divergent expression of MMP-2 between relapsing-remitting and secondary-progressive patients compared with controls was observed. Interferon-beta treatment was associated with significant suppression of MMP-9 and MMP-7 mRNA in relapsing-remitting patients, though no significant changes were observed in the secondary-progressive group. These results contribute to the understanding of the IFN-beta-mediated immunomodulatory and therapeutic effects in multiple sclerosis patients and also support evidence for distinct immune mechanism(s) underlying relapsing-remitting versus secondary-progressive multiple sclerosis. The study also suggests that MMPs may be considered as potential biomarkers for response to treatment as well as targets for immunotherapy in multiple sclerosis.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-alpha avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-alpha complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-alpha by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-alpha into ECM-FN. FN-bound TNF-alpha (FN/TNF-alpha) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-alpha activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Cheniotaxis through ECM gels in the presence of soluble or bound TNF-alpha was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-alpha required functional activated beta1 integrins, FN/TNF-alpha-induced MMP-9 secretion was independent of binding to beta1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing nAb to alpha4, alpha5, and beta1 subunits, which blocked cell adhesion; (2) a mAb that stimulated beta1 integrin-mediated adhesion; and (3) binding TNF-alpha to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-alpha-ECM interactions may serve to limit the cytokine's availability and bioactivity to target areas of inflammation.

Our objective was to find a possible correlation between telomerase activity, mean telomere length and human papillomavirus (HPV) presence and type in genital condylomata acuminata. Fifteen biopsies from women with genital condylomata acuminata and nine control tissue samples were tested for telomerase activity, mean telomere length, and HPV presence and type. All condylomata exhibited telomerase activity, compared to 78% of the control samples. The mean telomere length of condylomata was significantly (P < 0.002) shorter compared to telomere length in control tissue samples. All condylomata lesions were infected with HPV types 6/11, and more than half had additional infection with HPV 16/18. Mixed HPV 6/11 with 16/18 infection correlated with shorter telomeres than presence of HPV 6/11 alone in the lesions (4.68 ± 0.44 kb vs 4.97 ± 0.57 kb). None of the control tissue samples showed presence of HPV DNA. Telomerase activity may be a marker of proliferation rather than malignancy, whereas the mean telomere length could better serve as a marker for the progression of HPV lesions toward malignancy.

Several features suggest an immune mechanism operates in celiac disease. Information on the autoantibody repertoire in this condition is lacking. The purpose of the study was to investigate the reactivity of celiac patients sera to various autoantigens widely distributed in the human intestine. Seventy children, celiacs and controls, were evaluated for serum autoantibodies using ELISA and immunofluorescence. Celiac patients had increased prevalence of serum anti-single-stranded DNA (14%), anti-double-stranded DNA (23%), anti-cardiolipin (14%), and anti-endomysial autoantibodies (63%). The relevance of this finding on the extraintestinal manifestations of celiac disease or the coexistence of autoimmune conditions and celiac disease remains to be determined.

BACKGROUND Mechanisms whereby cells infected with human papillomavirus (HPV) escape immune surveillance, ultimately leading to invasive cervical carcinoma, may involve changes in local cytokine production, loss of responsiveness to cytokines, and alterations in the expression of immune-regulatory molecules such as histocompatibility-related leukocyte antigen (HLA) Class 1 and 2 and ICAM-I. This study examined the separate and combined effects of immune-activating cytokines, interferon (IFN)-γ, IFN-β, and tumor necrosis factor (TNF)-α, on the expression of these molecules. METHODS Membrane protein expression and cellular mRNA levels were analyzed in cervical carcinoma-derived cell lines, SiHa and CaSki (with low and high HPV16 copy number, respectively), after exposure to cytokines. RESULTS Both cell lines demonstrated constitutive expression of HLA Class 1 but not HLA Class 2 membrane antigens. IFN-γ and -β induced changes in Class 1 mRNA levels but not in membrane molecule expression. IFN-γ induced dose-dependent expression of Class 2 membrane and mRNA molecules in both cell lines (more pronounced in SiHa than in CaSki cells), which was antagonized by IFN-β. Constitutive ICAM-I membrane expression was observed only on CaSki cells, although ICAM-I mRNA was expressed in both cell lines. IFN-γ up-regulated the membrane expression of this molecule, whereas IFN-β led to its suppression. Differential modulation of ICAM-I mRNA was observed in both cell lines. A lack of response to TNF-α was observed throughout the experiments. CONCLUSIONS The findings of this study point to possible mechanisms leading to suppression of local immune response in the pathogenesis of HPV-associated neoplasia. They also emphasize the complexity of developing an efficient cytokine therapy for patients with premalignant cervical disease. Cancer 1997; 79:924-34. © 1997 American Cancer Society.

SUMMARY The αβ and γδ T cell receptor (TCR) repertoire in the peripheral blood and intestinal mucosa of six coeliac children and six age-matched controls was analysed by reverse transcription and polymerase chain reaction (PCR). No TCR αβ and γδ restriction was observed in coeliacs and controls. However, Vγ3 was expressed only in coeliac peripheral and intestinal T cells. Vδ2 was strongly expressed in coeliacs and scarcely transcribed in control cells. The unique expression of these γδ TCR in coeliac patients suggests that Vγ3 and perhaps Vδ2 TCR-bearing lymphocytes may play a role in the pathogenesis of coeliac disease.

Paget's bone disease developed in a patient with vitiligo. Scrupulous physical examination excluded further systemic or cutaneous involvement. The immunological workup revealed a reversed CD4/CD8 ratio due to a very low CD4 cell percentage and almost negligible responses to PHA as well as Con A, T cell mitogens. The pathogenic significance of these results, which point to phenotypic and functional T cell defects, is discussed.

We have postulated that in vivo autologous mixed lymphocyte reactions (AMLRs) are one mechanism in the development of the intrathyroidal lymphocytic infiltration of human autoimmune thyroid disease. Such a mechanism would explain the significant numbers of self-reactive T cells present in thyroid infiltrates as evidenced by cloning studies. However, infiltrating T cells in a variety of autoimmune disease including autoimmune thyroid disease, demonstrate bias in their use of T cell receptor (TcR) V gene families. In order to examine whether such TcR V gene bias may occur secondary to non-antigen specific in vivo AMLRs rather than secondary to specific autoantigen driven mechanisms we have examined the human TcR repertoire after prolonged AMLRs in vitro. Using 5 healthy donors in 1, 2 and 3 weeks AMLRs we showed stimulation indices of 3.1-6.5 after 3 weeks. The hTcR V alpha and V beta gene repertoire was assessed using the PCR technique and revealed an almost complete repertoire of V gene families at the beginning of the studies while at the end of 3 weeks a mean of only 5.2 V alpha genes were transcribed. Less restriction was seen in the hTcR V beta repertoire with a mean of 9 V beta genes used. These data demonstrate that the AMLR is able to mimic the marked bias in hTcR V gene family use seen within the inflammatory infiltrates of autoimmune diseases.

Transplantation involves preoperative ischemic periods that contribute to endothelial cell (EC) dysfunction and T-cell activation, leading to graft rejection. As hypoxia is a major constituent of ischemia, we evaluated its effect on the ability of ECs to express HLA-DR, which is required for presentation of antigens to T cells, and by itself serves as an important target for allogeneic T cells. Primary human umbilical vein ECs (HUVEC) and the human endothelial cell line EaHy926 were incubated in normoxia or hypoxia (PO(2)0.3%). Hypoxia increased the membranal expression (by 4-6 fold, P0.01) and secretion (by sixfold, P0.05) of HLA-DR protein, without influencing the accumulation of its mRNA. Alternative splicing, attenuated trafficking, or shedding from the plasma membrane were not observed, but the lysosomal inhibitor bafilomycin A1 reduced HLA-DR secretion. Hypoxia-induced endothelial HLA-DR elevated and diminished the secretion of IL-2 and IL-10, respectively, from co-cultured allogeneic CD4(+) T cells in a HLA-DR-dependent manner, as demonstrated by the use of monoclonal anti-HLA-DR. Our results indicate a yet not fully understood post-translational mechanism(s), which elevate both membranal and soluble HLA-DR expression. This elevation is involved in allogeneic T-cell activation, highlighting the pivotal role of ECs in ischemia/hypoxia-associated injury and graft rejection.

The effects of human prolactin on enriched peripheral B lymphocytes obtained from healthy males were examined. Immunoregulation by prolactin was studied in B cells activated with either anti-IgM alone, or anti-IgM antibodies and recombinant interleukin-2 (r-IL-2), as well as control resting B cells. Expression of IL-2 receptors (IL-2R), and IgM and IgG in the culture supernatants were used as a measure of B cell activation and differentiation. Prolactin significantly synergized with IL-2 in the enhancement of surface expression of IL-2R on anti-IgM treated B cells. Although no differentiating effect was observed on resting B cells, prolactin (0.2–100 ng/ml) exhibited a dose-dependent enhancement upon both IgM and IgG secretion from B cells treated with anti-IgM and IL-2. In the absence of exogenously added IL-2 similar differentiating effect were observed in B cells treated with anti-IgM at prolactin concentrations of 0.2–10 ng/ml, but not 100 ng/ml. Thus, the present results demonstrate the modulatory effect of prolactin on activation and differentiation of anti-IgM triggered human B cells, and emphasize the importance of co-stimulatory signal mediated by IL-2 in B cell responses to high prolactin levels. These findings extend the immunoregulatory effects of prolactin, previously confirmed for T cells, to the B cell arm of the immune response, and suggest an important role of prolactin in mediating adaptation and communication between the nerve and immune systems.

Interleukin-10 is an anti-inflammatory cytokine and consequently is considered by many to have a protective role in heart failure, as opposed to the notorious tumor necrosis factor-alpha.To test the hypothesis of the possible beneficial impact of IL-10 on mortality in systolic heart failure patients in relation to their circulating TNFalpha levels.We measured circulating levels of IL-10 and TNFalpha in 67 ambulatory systolic heart failure patients (age 65 +/- 13 years).Mortality was or tended to be higher in patients with higher levels (above median level) of circulating TNFalpha (9/23, 39% vs. 6/44, 14%; P = 0.02) or IL-10 (10/34, 30% vs. 5/33, 15%; P = 0.10). However, mortality was highest in the subset of patients with elevation of both markers above median (7/16, 44% vs. 8/51, 16%; P = 0.019). Elevation of both markers was associated with more than a threefold hazard ratio for mortality (HR 3.67, 95% confidence interval 1.14-11.78).Elevated circulating IL-10 levels in systolic heart failure patients do not have a protective counterbalance effect on mortality. Moreover, patients with elevated IL-10 and TNFalpha had significantly higher mortality, suggesting that the possible interaction in the complex inflammatory and anti-inflammatory network may need further study.

SUMMARY Plasma levels of biologically active IL-1, tumour necrosis factor (TNF) and IL-6 were measured before, during and after coronary artery bypass graftings (CABG) (n= 9) and cholecystectomy (CHO. n= 9), and in normal controls (nine healthy volunteers). Mean pre-operative IL-1 concentration in four of the nine CABG patients was 0.452 ± 0.03 ng/ml, significantly (P < 0.001) higher than that of the other five (0.045 ± 0.009 ng/ml), CHO patients (0.035 ± 0.005 ng/ml) and controls (0.029 ± 0.008 ng/ml). Three of the four patients with high pre-operative IL-1 had functional capacity IV, while the other five had functional capacity IIa or IIb. Slight IL-1 elevation after anaesthesia, followed by reduction after initiation of bypass, elevation on completion of surgery and reduction to basal levels after 7 days was found in patients undergoing CABG. Mean basal TNF levels of CABG and CHO patients did not differ, but were higher than those of controls (2.85 ± 0.5 ng/ml for CABG, 2.05 ± 0.06 ng/ml for CHO, 0.72 ± 007 ng/ml for normals, P < 0.001). A unique kinetics of release during CABG was observed also for TNF. Mean pre-operative IL-6 levels were normal (50 ± 3 ng/ml for CABG. 50 ± 0.5 ng/ml for CHO and 65 ± 10 ng/ml for controls). Gradual elevation to a mean peak of 725 ± 100 ng/ml on completion of CABG was observed as compared with 275 ± 50 ng/ml in CHO (P < 0.01). On the seventh post-operative day mean IL-6 levels returned to normal. Two patients with post-operative low-grade fever (38°C) had high, late eytokine levels. One of these two patients had leucocytosis, sterile discharge from the operative wound and was diagnosed as suffering from the Dressler syndrome. In this study elevated cytokine values and unique kinetics of release into the serum were found in patients undergoing CABG.

Apparently complex modulatory effects of alpha-interferon (alpha-IFN), tumor necrosis factor (TNF), and epidermal growth factor (EGF) have been found in neoplastic human thyroid cells, which could possibly affect the final outcome in neoplastic disease. This was achieved by examining the influence of alpha-IFN, TNF, and EGF alone and in combination, on human leukocyte antigen-DR (DR) antigen expression and viability of neoplastic and non-neoplastic human thyroid cells in culture. alpha-IFN-induced DR antigen expression on non-neoplastic human thyroid cells, whereas TNF-alpha or EGF alone were ineffective. The addition of the same TNF-alpha concentrations (10 to 100 ng/ml) to alpha-IFN enhanced the expression of DR antigens compared with the effect of alpha-IFN alone. However, EGF inhibited alpha-IFN-induced DR on the same cells and at the same concentrations (10 to 500 ng/ml) at which the growth factor alone was ineffective. In contrast to the common pattern of cytokine effects on DR expression of all nonmalignant thyroid cell lines, neoplastic thyroid cell lines showed divergent responses to alpha-IFN, TNF-alpha, and EGF. In three malignant thyroid cell lines that were DR negative (follicular carcinoma WRO 82-1 and NRO 87-1 cell lines, and anaplastic carcinoma ARO 81-1), DR antigen could be induced by alpha-IFN and enhanced by TNF-alpha, whereas EGF was ineffective. In a fourth cell line (an anaplastic carcinoma SW1736) alpha-IFN, TNF-alpha, and EGF alone were capable of inducing DR, and a combination of either TNF-alpha and EGF with alpha-IFN potentiated DR induction. In a fifth neoplastic cell line (papillary carcinoma, NPA) that constitutively expressed surface DR, its expression was inhibited by both alpha-IFN and TNF-alpha and was not affected by EGF.

We have used non-autoimmune non-neoplastic human thyroid cells to explore the role of surface class I and DR antigens on these cells' sensitivity towards T and Natural Killer (NK) cell cytotoxicity. Non-treated thyrocytes expressed class I but no DR antigens. Following incubation with gamma-interferon (gamma-IFN) class I antigens were markedly elevated and DR expression was induced. Whereas non-treated thyrocytes were minimally lysed by sensitized T cells, they served as appropriate targets for NK cells. Following incubation with gamma-IFN, the thyroid cells became highly sensitive to T cell lysis, with no significant reduction in their vulnerability to NK cell killing. The addition of monoclonal anti class I or DR antigens, or brief acid treatment which specifically eliminates class I molecules, inhibited T cell cytotoxicity but enhanced the sensitivity to lysis by NK cells. Thus, the presence of HLA antigens on the same thyroid cells have an opposite effect on two major cytotoxi mechanisms. Our findings are relevant within the context of recent suggestions of intervening with target HLA antigens for the management of autoimmune and malignant diseases.

SUMMARYWe studied B cells from 18 patients with B-CLL, six of them with autoimmune haemolytic anaemia, for spontaneous secretion of IL-6. Our aim was to determine whether the increased incidence of autoimmune disease found in B-CLL patients is associated with enhanced spontaneous IL-6 secretion. IL-6 was measured by the effect of B cell supernatants on the proliferation of an IL-6 dependent plasmacytoma cell line T1165. The highest IL-6 values (7.4±1.8 U/ml) were measured in supernatants derived on day 3 of culture from lymphocytes of the six patients with B-CLL and concomitant autoimmune disease. The maximal IL-6 values for 10 patients with B-CLL only were 2.8±0.3 U/ml and for 10 age-matched controls, 0.8±0.3 U/ml (P < 0.01, each group compared with the other). We conclude that there is an association between B-CLL, autoimmune disease and the spontaneous in vitro secretion of IL-6. Further studies are needed to determine whether the IL-6 secretion plays a role in the pathogenesis of autoimmune disease in patients with B-CLL.

Interleukin-2 (IL-2) production was studied in T lymphocytes and isolated CD4+ T lymphocytes from 12 patients with primary fibromyalgia syndrome and 10 healthy volunteers. The dose and time kinetics of IL-2 production by concanavalin A-stimulated T cells and CD4+ T cells of fibromyalgia patients differed from findings in controls by 1) a need for a higher concentration of mitogen in order to achieve optimal IL-2 secretion, and 2) a delay in the peak time of optimal IL-2 secretion. Unlike normal IL-2 secretion, which was higher after removal of CD8+ T cells, the pattern and degree of IL-2 secretion by cells from fibromyalgia patients were not changed following removal of CD8+ T cells. Addition of calcium ionophore in assays using suboptimal concanavalin A concentrations did not correct the reduction in IL-2 secretion by fibromyalgia patient T cells, but addition of phorbol myristate acetate induced normal secretion of IL-2. These findings suggest that there is a defect in the IL-2 pathway, which is related to protein kinase C activation and does not involve impairment of Ca2+ elevation, in patients with fibromyalgia.

Cytokines are important mediators of the inflammatory reaction and microvascular injury after trauma and tissue ischemia. The plasma activity of a cytokine reflects the net effect of positive and negative signals. We examined the sequential serum activity of IL-1, IL-2, IL-6, and TNF in a severe model of splanchnic artery occlusion (SAO) shock induced in rats by total occlusion of the superior mesenteric and the celiac arteries for 40 min. A control group with negligible surgical intervention and two sham-shock groups, one with minor operation and another with major surgery employed in SAO rats, both without vascular occlusion, were also studied. No IL-1 activity was detected throughout the 190-min experimental protocol in any of the groups. Low activity of IL-2 was measured only in SAO rats (approximately 1 U/ml at the peak). We found graded increases in serum TNF and IL-6 activities which were proportional to the surgical trauma and were highest in SAO rats (IL-6 up to 30 U/ml, P less than 0.01 from both sham groups; TNF, 2500 pg/ml 30 min after reperfusion, P less than 0.01 from both sham groups). These data further support the role played by cytokines in the early mediation of surgical trauma and shock.

A diabetic patient is described presenting psoriasis, necrobiosis lipoidica diabeticorum, granuloma annulare, and vitiligo and with a history of recurrent erysipelas and mycotic infections. Scrupulous physical examination excluded further systemic or cutaneous involvement. The immunological workup revealed both phenotypic and functional defects in cellular immunity.

Human autoimmune thyroid cells "spontaneously" express MHC-class II antigens. These antigens have been assumed to trigger thyroid-specific helper T cell clones, leading in turn to the expansion of thyroid autoantibody-secreting B cells. Thyroid cells derived from non-autoimmune subjects do not express MHC-class antigens, but these can be readily induced with gamma-interferon. We have addressed the issue of whether it is sufficient for normal thyroid cells to bear class II antigens in order to trigger autologous T cells. We found that non-autoimmune thyrocytes expressing DR antigens fail to stimulate autologous resting T cells. However, proliferative activity and interleukin-2 secretion were observed when fresh T cells were first triggered by autologous non-T cells and then incubated with thyrocytes. More CD8 than CD4 cells proliferated in the T:thyrocyte cultures, but CD4 cells were necessary for the proliferation and interleukin-2 secretion. Addition of antibodies to thyroglobulin or to DR antigens inhibited T cell proliferation and interleukin-2 secretion, thus pointing to T cell recognition of both thyroid-specific and DR antigens. Evaluation of the function of the thyroid stimulated T cells revealed very potent suppressor but negligible helper and cytotoxic activities. It would seem, therefore, that DR-restricted T cell activation by autologous antigen on non-autoimmune thyroid cells does occur, but since it results in enhanced suppression, its nature seems protective, thus leading to maintenance of immunological self-tolerance.

Monocytes remodel the extracellular matrix (ECM) by secreting proteins composing the ECM such as fibronectin (FN) and degrading proteases such as matrix metalloproteinase-9 (MMP-9), which cleaves FN into fragments. The effects of FN and its fragmented products on the expression of monocyte MMP-9 are controversial and largely unknown. We showed that in human monocytes, the proinflammatory cytokine TNF-α induced MMP-9 secretion and increased fragmentation of FN into distinct fragments. When primary monocytes or the U937 monocytic cell line were incubated on a plastic substrate, plastic-coated with native FN, and plastic-coated with fragmented FN (frag-FN), native FN inhibited TNF-α-induced proMMP-9 secretion by twofold (P

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase-9 (MMP-9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor α (TNF-α)]. We show that hypoxia (

Exposure of endothelial cells (ECs) to hypoxia has separately been shown to induce their angiogenesis or death. Matrix metalloproteinase (MMP)-2 is associated with EC angiogenesis, although recent studies also implicate this molecule in EC death. We studied the effect of hypoxia in the absence or presence of TNF-α (characteristic of the inflammatory microenvironment accompanying hypoxia) on MMP-2 expression and its role in angiogenesis (proliferation, migration, and tube formation) and in the death of primary human umbilical vein endothelial cells (HUVECs). Hypoxia alone (24–48 h in 0.3% O2in the hypoxic chamber) and furthermore, when combined with TNF-α, significantly enhanced MMP-2 expression and activity. Hypoxia also led to a reduction in membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 mRNA and protein while enhancing the expression of αvβ3integrin and the cytoskeletal protein phosphopaxillin. Moreover, hypoxia led to colocalization of αvβ3and MMP-2, but not MT1-MMP, with phosphopaxillin in ECs. These results suggest MT1-MMP-independent activation of MMP-2 during hypoxia and support interactions between the ECM, integrins, and the cytoskeleton in hypoxia-induced MMP-2-related functions. Hypoxia enhanced EC migration in an MMP-2-dependent manner while leading to a reduction of cell number via their apoptosis, which was also dependent on MMP-2. In addition, hypoxia caused an aberrant tubelike formation on Matrigel that appeared to be unaffected by MMP-2. The hypoxia-induced, MMP-2-dependent migration of ECs is in accordance with the proangiogenic role ascribed to MMP-2, while the involvement of this protease in the hypoxia-related death of ECs supports an additional apoptotic role for this protease. Hence, in the hypoxic microenvironment, MMP-2 appears to have a dual autocrine role in determining the fate of ECs.

Whereas the diagnosis of classical pertussis has traditionally been based on clinical criteria, increasing numbers of atypical presentations suggest the need for an extensive laboratory-based approach.To assess the relative efficacy of clinical and laboratory methods in the diagnosis of Bordetella pertussis by patient age and immunization status.We compared the clinical and laboratory diagnosis of B. pertussis in 87 pre-vaccinated, 78 recently vaccinated, and 75 post-vaccinated children with suspected pertussis. Serum and nasopharyngeal swabs were obtained for serology, culture and polymerase chain reaction.PCR and culture identified 41% and 7% of patients with B. pertussis, respectively (P0.001). All positive cultures were PCR-positive. Positive PCR was less common among those recently vaccinated than among those in the pre- (P0.001) and post-vaccinated groups (P0.05). Positive culture was more common among those pre-vaccinated than among those recently vaccinated (P0.01). Positive tests for immunoglobulin M and A were more common among the post-vaccinated than the pre- and recently vaccinated (P0.001), respectively. Logistic regression analyses revealed that clinical criteria have no significant association with infection in recently and post-vaccinated children. Among the pre-vaccinated children, whoop and cough duration were associated with a positive PCR (odds ratio 7.66 and 0.5, P0.001). Seventy-six percent of pre-vaccinated, 39% of recently vaccinated and 40% of post-vaccinated children with positive PCR did not meet the U.S. Centers for Disease Control diagnostic criteria for B. pertussis.PCR is a useful tool for pertussis diagnosis, particularly in pre-vaccinated infants. The yield of culture and serology is limited, especially among pre- and recently vaccinated children. In pre-vaccinated infants with whoop and less than 2 weeks of cough, PCR testing should be implemented promptly.

Our objective in this study was to correlate immunosuppressive properties of follicular fluid (FF) and media conditioned by zygotes and early embryos with the occurrence of conception in an in vitro fertilization (IVF) program.Fifty-seven IVF patients were studied. Donor lymphocytes were incubated with mitogens and FF from mature oocytes or conditioned media from zygotes and early embryos. Proliferation was assessed by radioactive thymidine incorporation. Proliferation Index (PI) was the ratio between radioactive labeling of lymphocytes incubated in the presence and absence of a mitogen.The FFs and media conditioned by zygotes from conception cycles had higher immunosuppressive activity than those from non-conception cycles.Immunosuppressive activity present in FF and media conditioned by zygotes may be a major determinant of conception in IVF, and may serve as a marker for embryo quality.