BACKGROUND

Multiple sclerosis is a demyelinating disease mostly of autoimmune origin that affects and damages the central nervous system, leading to a disabling condition. The aim of the present study was to investigate whether administration of mesenchymal stem cells from human periodontal ligament (hPDLSCs) could ameliorate multiple sclerosis progression by exerting neuroprotective effects in an experimental model of autoimmune encephalomyelitis (EAE).

METHODS

EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG)<em>35</em>-55 in C57BL/6 mice. After immunization, mice were observed every 48 hours for signs of EAE and weight loss. At the onset of disease, approximately 14 days after immunization, EAE mice were subjected to a single intravenous injection of hPDLSCs (10(6) cells/150 μl) into the tail vein. At the point of animal sacrifice on day 56 after EAE induction, spinal cord and brain tissues were collected in order to perform histological evaluation, immunohistochemistry and western blotting analysis.

RESULTS

Achieved results reveal that treatment with hPDLSCs may exert neuroprotective effects against EAE, diminishing both clinical signs and histological score typical of the disease (lymphocytic infiltration and demyelination) probably through the production of neurotrophic factors (results focused on brain-derived neurotrophic factor and nerve growth factor expression). Furthermore, administration of hPDLSCs modulates expression of inflammatory key markers (tumor necrosis factor-α, interleukin (IL)-1β, IL-10, glial fibrillary acidic protein, Nrf2 and Foxp3), the release of CD4 and CD8α T cells, and the triggering of apoptotic death pathway (data shown for cleaved caspase 3, p53 and p21).

CONCLUSIONS

In light of the achieved results, transplantation of hPDLSCs may represent a putative novel and helpful tool for multiple sclerosis treatment. These cells could have considerable implication for future therapies for multiple sclerosis and this study may represent the starting point for further investigations.

This phase I clinical trial (NCT019<em>35</em>843) is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell (CART) immunotherapy targeting human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancers (BTCs) and pancreatic cancers (PCs). Eligible patients with HER2-positive (>50%) BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nab-paclitaxel (100-200 mg/m2) and cyclophosphamide (15-<em>35</em> mg/kg). CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CART-HER2 cell infusion (median CAR+ T cell 2.1 × 106/kg). The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgia/arthralgia, and lymphopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase (>9 ULN), adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastrointestinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1-2 delayed fever, accompanied by the release of C-reactive protein and <em>interleukin</em>-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months (range, 1.5-8.3 months). Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encouraging signals of clinical activity.

BACKGROUND

Neurocognitive dysfunction occurs frequently after open-heart surgery. Cerebral microembolization, inflammation, blood-brain barrier (BBB) dysfunction, and impaired cerebral oxygenation are considered among possible etiologies. The relationships between intraoperative microembolic signals and the release of cerebrospinal fluid (CSF) markers of inflammation, neuronal and glial cell injuries, and BBB function were evaluated after cardiac surgery with cardiopulmonary bypass.

METHODS

Ten patients undergoing aortic valve replacement were included. The CSF was obtained the day before and 24 hours after surgery for assessment of neuronal damage (neuron-specific enolase, total tau, and neurofilament light chain protein), glial cell injury (S-100B, glial fibrillary acidic protein), BBB integrity (CSF to serum albumin ratio) and cytokines (interleukin-6, interleukin-8). Intraoperative extent of microemboli and their occurrence were described using the transcranial Doppler technique.

RESULTS

Intraoperatively, 354±79 microemboli were detected; 81% after release of the aortic cross clamp. The S-100B and glial fibrillary acidic protein increased by 35% (p<0.01) and 25% (p=0.055), respectively. Neuron-specific enolase, total tau, and neurofilament light chain protein, were not significantly affected by the surgery. The CSF albumin increased by 13% (p<0.05) while serum albumin decreased by 27% (p<0.0001). Thus, CSF to serum albumin ratio increased by 61% (p=0.011). There was a 3.5- and 12-fold increase in interleukin-6 (p<0.001) and interleukin-8 (p<0.05), respectively. Microembolic signals did not correlate to changes in CSF glial injury markers, the CSF to serum albumin ratio, or CSF cytokines.

CONCLUSIONS

Cardiac surgery with cardiopulmonary bypass causes cerebral inflammation, glial cell injury, and BBB dysfunction without biochemical signs of neuronal damage. These changes are not associated with intraoperative microembolization.

OBJECTIVE

To identify biomarkers which distinguish severe sepsis/septic shock from uncomplicated sepsis in the Emergency Department (ED).

METHODS

Patients with sepsis underwent serial blood sampling, including arrival in the ED and up to three subsequent time points over the first 24 hours. Messenger RNA (mRNA) levels of 13 genes representing arms of the innate immune response, organ dysfunction or shock were measured in peripheral blood leucocytes using quantitative PCR, and compared with healthy controls. Serum protein concentrations of targets differentially expressed between uncomplicated sepsis and severe sepsis/septic shock were then measured at each time point and compared between the two patient groups.

RESULTS

Of 27 participants (median age 66 years, (IQR <em>35</em>, 78)), 10 had uncomplicated sepsis and 17 had sepsis with organ failure (14 septic shock; 3 had other sepsis-related organ failures). At the time of first sample collection in the ED, gene expression of <em>Interleukin</em> (IL)-10 and Neutrophil Gelatinase Associated Lipocalin (NGAL) were significantly higher in severe sepsis than uncomplicated sepsis. Expression did not significantly change over time for any target gene. Serum concentrations of IL-6, IL-8, IL-10, NGAL and Resistin were significantly higher in severe sepsis than uncomplicated sepsis at the time of first sample collection in the ED, but only IL-8, NGAL and Resistin were consistently higher in severe sepsis compared to uncomplicated sepsis at all time points up to 24 h after presentation.

CONCLUSIONS

These mediators, produced by both damaged tissues and circulating leukocytes, may have important roles in the development of severe sepsis. Further work will determine whether they have any value, in addition to clinical risk parameters, for the early identification of patients that will subsequently deteriorate and/or have a higher risk of death.

OBJECTIVE

To determine the contribution of soft-tissue trauma plus hemorrhage, bone fracture and hemorrhage, as well as the contribution of bone fracture, soft-tissue trauma and hemorrhage on host immune function.

METHODS

Adult male mice (n = 6/group).

METHODS

Prospective, randomized, controlled study.

METHODS

Animal laboratory at a university-affiliated hospital.

METHODS

Closed-bone fracture (right lower leg; external fixation) and/or soft-tissue trauma (2.5-cm midline laparotomy, closed in two layers) were induced before hemorrhagic shock (mean arterial blood pressure of <em>35</em> +/- 5 (SEM) mm Hg for 90 mins, followed by fluid resuscitation) in male C3H/HeN mice and the animals were killed at 72 hrs after initiation of the experiment.

RESULTS

Splenocyte interleukin (IL)-2 and IL-3 release capacity, as well as splenic and peritoneal macrophage IL-1 and IL-6 release capacity were determined. Different traumatic insults, i.e., bone fracture or soft-tissue trauma in conjunction with hemorrhage, produced comparable immune depression. More significant depression of splenocyte IL-2 and IL-3 release capacity as well as macrophage IL-1 and IL-6 release capacity occurred with the combined insult (i.e., bone fracture/soft-tissue injury and hemorrhage) than after bone injury or tissue trauma alone with hemorrhage.

CONCLUSIONS

The combination of closed-bone fracture and soft-tissue trauma before hemorrhage leads to even more compromised immunity than either soft-tissue trauma or closed-bone fracture along with hemorrhage. The markedly depressed immune function following bone injury, soft-tissue trauma, and hemorrhagic shock may contribute to the increased susceptibility of severely injured patients to sepsis and the ensuing multiple organ failure in the clinical situation.

In the process of homing, CD34(+) hematopoietic progenitor cells migrate across the bone marrow endothelium in response to stromal cell-derived factor (SDF)-1. To develop more efficient stem cell transplantation procedures, it is important to define the adhesion molecules involved in the homing process. Here, we identified the adhesion molecules that control the migration of primary human CD34(+) cells across human bone marrow endothelial cells. Migration of CD34(+) cells is enhanced across <em>interleukin</em> 1beta prestimulated bone marrow endothelium, suggesting an important role for the endothelium in adhesion and formation of the chemotactic gradient. Under these conditions, 30-100 ng/ml SDF-1 induced a rapid and efficient migration of CD34(+) cells (+/- 46% migration in 4 h). In contrast, 600-1,000 ng/ml SDF-1 were required for optimal migration across fibronectin-coated filters. Subsequent studies revealed that transendothelial migration of CD34(+) cells is mediated by beta1- and beta2-integrins and PECAM-1 (CD31) but not by CD34 or E-selectin. Whereas these antibodies individually blocked migration for 25%-<em>35</em>%, migration was reduced by 68% when the antibodies were combined. Thus, these adhesion molecules play specific and independent roles in the transmigration process. Finally, O-glycosylated proteins appeared to play a role, since SDF-1-induced migration of CD34(+) cells (treated with a glycoprotease from Pasteurella haemolytica) across endothelial cells was clearly inhibited. In conclusion, we show that efficient SDF-1-induced migration of primary human CD34(+) cells across bone marrow endothelium is mediated by beta1-integrins, beta2-integrins, CD31 and O-glycosylated proteins.

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein-Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed-Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce <em>interleukin</em> (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (31 of 37, 84 per cent) as compared with LMP1-negative HD cases (<em>35</em> of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD.

We studied the role of <em>interleukin</em>-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) in the proliferative response of transformed human renal interstitial fibroblast cell lines established from either a kidney with glomerulonephritis and interstitial fibrosis or a normal kidney in comparison to primary human foreskin fibroblasts. Growth of fibrosis-derived renal fibroblasts was inhibited in the presence of IL-1 receptor antagonist (IL-1Ra) by <em>35</em>% (P < 0.005), suggesting that these cells produce IL-1 and possess IL-1 receptors as part of paracrine growth. In contrast, spontaneous proliferation of fibroblasts derived from a normal kidney or normal skin were not inhibited by IL-1Ra. In fibrosis-derived but not in normal renal cells, fibronectin synthesis was increased 2.2-fold (P < 0.01) in the presence of IL-1Ra. Addition of exogenous IL-1 beta or bFGF stimulated proliferation of skin fibroblasts. In contrast, growth of fibrosis-derived renal fibroblasts was stimulated by IL-1 beta and unchanged by bFGF. Growth of normal kidney fibroblasts was unaffected by bFGF and inhibited by IL-1 beta. We conclude that compared to normal fibroblasts, fibrosis-derived renal fibroblasts have a different cytokine-response profile, are IL-1-dependent, produce IL-1 as a paracrine growth factor and do not proliferate to bFGF, a classical fibroblast growth factor.

We demonstrate here that the CC chemokines macrophage inflammatory protein-3alpha (MIP-3alpha), macrophage inflammatory protein-3beta (MIP-3beta) and the CX3C chemokine fractalkine induce the chemotaxis of <em>interleukin</em>-2 (IL-2)-activated natural killer (IANK) cells. In addition, these chemokines enhance the binding of [gamma-<em>35S</em>]guanine triphosphate ([gamma-<em>35S</em>]GTP) to IANK cell membranes, suggesting that receptors for these chemokines are G protein-coupled. Our results show that MIP-3alpha receptors are coupled to Go, Gq and Gz, MIP-3beta receptors are coupled to Gi, Gq and Gs, whereas fractalkine receptors are coupled to Gi, and Gz. All three chemokines induced a robust calcium response flux in IANK cells. Cross-desensitization experiments show that MIP-3alpha, MIP-3beta or fractalkine use receptors not shared by each other or by the CC chemokine regulated on activation, normal, T-cell expressed, and secreted (RANTES), the CXC chemokines stromal-derived factor-1alpha (SDF-1alpha) and interferon-inducible protein-10 (IP-10), or the C chemokine lymphotactin.

The value of coronary artery calcium (CAC) scoring versus multiple biomarkers in increasing risk prediction for cardiovascular disease (CVD) remains unknown. The study group consisted of 1,286 asymptomatic participants (mean ± SD 59 ± 8 years old) with no known coronary heart disease. Mean follow-up time was 4.1 ± 0.4 years with the primary outcome of combined CVD (cardiac death, myocardial infarction, stroke, and late target vessel revascularization). CAC was calculated by the method of Agatston. Biomarkers measured were C-reactive protein, <em>interleukin</em>-6, myeloperoxidase, B-type natriuretic peptide, and plasminogen activator-1. During follow-up <em>35</em> participants developed CVD events including cardiac deaths (6%), myocardial infarction (23%), strokes (17%), and late revascularizations (54%). In Cox proportional-hazards models adjusted for Framingham Risk Score (FRS), presence of log CAC beyond FRS was associated with increased hazards for CVD events (hazard ratio 1.7, 95% confidence interval [CI] 1.4 to 2.0, p <0.001). Multiple biomarkers score was also associated with increased risk beyond FRS (hazard ratio 2.1, p = 0.02) per 1-U increase in score; however, the c-statistic did not increase significantly (0.75, 95% CI 0.68 to 0.84, p = 0.32). The c-statistic increased when log CAC was incorporated into FRS without or with multiple biomarkers score (c-statistic 0.84, p = 0.003 and p = 0.008 respectively). Addition of CAC to risk factors showed significant reclassification (net reclassification improvement 0.<em>35</em> (95% CI 0.11 to 0.58, p = 0.007; integrated discrimination index 0.076, p = 0.0001), whereas addition of multiple biomarkers score did not show significant reclassification. In conclusion, in this study of asymptomatic subjects without known CVD, addition of CAC but not biomarkers substantially improved risk reclassification for future CVD events beyond traditional risk factors.

Regulatory T cells come in many different forms depending on their mode of action or developmental origin. Data now show that <em>interleukin</em> <em>35</em>, an immunomodulatory cytokine secreted by regulatory T cells, and <em>interleukin</em> 10 induce so-called 'iTR<em>35</em> cells', which may have an important role in the phenomenon of infectious tolerance.

OBJECTIVE

Low organisational justice has been shown to be associated with increased risk of various health problems, but the underlying mechanisms remain unclear. We tested whether organisational injustice contributes to chronic inflammation in a population of middle-aged men and women.

METHODS

This prospective cohort study uses data from 3205 men and 1204 women aged <em>35</em>-55 years at entry into the Whitehall II study (phase 1, 1985-1988). Organisational justice perceptions were assessed at phase 1 and phase 2 (1989-1990) and circulating inflammatory markers C-reactive protein (CRP) and <em>interleukin</em> (IL)-6 at phase 3 (1991-1993) and phase 7 (2003-2004).

RESULTS

In men, low organisational justice was associated with increased CRP levels at both follow-ups (phase 3 and 7) and increased IL-6 at the second follow-up (phase 7). The long term (phase 7) associations were largely independent of covariates, such as age, employment grade, body mass index and depressive symptoms. In women, no relationship was found between organisational justice and CRP or IL-6.

CONCLUSIONS

This study suggests that organisational injustice is associated with increased long-term levels of inflammatory markers among men.

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31-<em>35</em>% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and <em>interleukin</em>-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

BACKGROUND

Few studies are available to compare the potential benefits of natural orifice transluminal endoscopic surgery (NOTES) approaches to traditional surgery.

OBJECTIVE

To compare complications, surgical stress, and postoperative pain.

METHODS

Prospective study in dogs.

METHODS

Research laboratory.

METHODS

Thirty dogs.

METHODS

Oophorectomy procedures were performed via NOTES and laparoscopic and traditional open surgery.

METHODS

Operative time, pain scores, systemic stress parameters (cortisol, glucose), surgical stress markers (interleukin 6, C-reactive protein), 3-day observation.

RESULTS

Median operative times were 76, 44, and 35 minutes for the NOTES, laparoscopic, and open procedures, respectively, with the NOTES procedure being significantly longer than the other 2 procedures. All ovaries were completely excised, and all the animals survived without complications. The NOTES animals had greater increases in serum cortisol concentrations at 2 hours but no statistically significant differences in glucose concentrations compared with the other groups. Serum interleukin 6 and C-reactive protein concentrations were significantly increased at specific times compared with baseline in the NOTES group, but not in the open or laparoscopic surgery groups. Based on the cumulative pain score and nociceptive thresholds, the animals in the NOTES group demonstrated less evidence of pain.

CONCLUSIONS

Small sample size, limited follow-up.

CONCLUSIONS

Although the NOTES oophorectomy procedures took approximately twice as long and there may be more evidence of tissue damage as judged by increases in serum cortisol and interleukin 6 concentrations, the dogs in the NOTES group had lower pain scores, especially when compared with animals undergoing open surgery.

The cytokine <em>interleukin</em>-2 is a primary modulator of the immune response that occurs after infection, trauma, and transplant rejection, yet its role as a mediator of associated metabolic changes in surgical illness is unknown. We studied clinical and metabolic responses in eleven tumor-bearing humans with normal renal and hepatic function receiving bolus intravenous (I.V.) <em>interleukin</em>-2 (30,000 U/kg). Additional subjects (n = 6) were pretreated with the cyclooxygenase inhibitor, ibuprofen (1600 mg, orally), before <em>interleukin</em>-2 administration. Serial measurements were made of vital signs, symptoms, hematology, and plasma concentrations of pituitary and stress hormones and selected cytokines. Administration of <em>interleukin</em>-2 resulted in fever, tachyacardia, "flu-like" symptoms, and neurohormonal elaboration. The responses observed were quantitatively similar to those that occurred after endotoxin administration in healthy subjects (n = 13), but differed in the following manner: 1) the onset of fever and endocrine changes occurred after a longer latent interval (180-240 minutes vs. 60-90 minutes after endotoxin), 2) peak responses after the administration of <em>interleukin</em>-2 also occurred later, 3) no increased circulating tumor necrosis factor was detected after administration of <em>interleukin</em>-2 (peak plasma concentration was greater than <em>35</em> pg/ml vs. 270 +/- 70 pg/ml after endotoxin administration), and 4) administration of <em>interleukin</em>-2 but not of endotoxin was associated with increased circulating concentrations of gamma interferon (peak plasma concentration 1.7 +/- 0.2 NIH U/ml vs. less than 0.1 NIH U/ml after endotoxin administration). Fever and neurohormonal responses after <em>interleukin</em>-2 administration were greatly attenuated by ibuprofen administration. <em>Interleukin</em>-2 induces other cytokines that exert their effects largely through the cyclooxygenase pathway. <em>Interleukin</em>-2 may be an important signal, initiating the integrated host responses to infection and injury.

Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using <em>35</em> k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the <em>interleukins</em> IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).

BACKGROUND

We wished to determine whether the addition of statins affect cardiovascular events and markers of inflammation in patients with heart failure.

RESULTS

A total of 446 patients with heart failure and ejection fraction < or =<em>35</em>% were followed in a prospective, nonrandomized fashion and were classified according to treatment with a statin. We determined all-cause mortality, cardiovascular morbidity, and serum markers of inflammation over a 24-month period. Statin therapy in patients with heart failure was associated with decreased all-cause mortality at 2 years compared with those not on statin therapy (15% versus 33%, P < .005) as well as hospitalizations for heart failure (22% versus 38%, P = .001) and nonfatal myocardial infarction (11% versus 15%, P < .001). In addition, statin therapy was associated with a decrease in serum levels of C-reactive protein (1.12 +/- 0.13 versus 1.47 +/- 0.11 mg/dL, P = .001), <em>interleukin</em>-6 (13.3 +/- 0.8 versus 17.3 +/- 1.4 ng/dL, P = .001), and tumor necrosis factor-alpha receptor II (24.3 +/- 1.0 versus 34.5 +/- 3.0 ng/dL, P = .001).

CONCLUSIONS

The use of statin therapy in this nonrandomized trial was associated with a significant reduction in all-cause mortality and cardiac morbidity. In addition, the improvement in levels of several serum inflammatory markers with statin therapy suggests in part possible mechanisms by which these agents may exert their benefits.

To identify T-helper (Th)-cell epitopes, we analyzed 25 synthetic peptides, which included most of the 495-amino-acid sequence of the envelope (E)-glycoprotein of dengue 2 virus. The peptides were analyzed in three mouse strains, BALB/c (H-2d), C57BL/6 (H-2b), and outbred NIH-Swiss, for their ability to elicit antibody or prime the Th-cell compartment following two inoculations in Freund's incomplete adjuvant. Sixteen peptides were able to elicit an antipeptide antibody response in one or more mouse strain. Eleven antipeptide serum pools were able to bind to virus in ELISA. Fifteen peptides primed one or more haplotype for an in vitro antipeptide Th-cell response as measured by blastogenesis. Th-cell activation was generally confirmed by measurable in vitro production of <em>interleukin</em> (IL)-2/IL-4. Nine peptides that were positive for in vitro blastogenesis, 1-2, <em>35</em>, 4-6, 79, 142, 208, 06, 16, and 17, elicited virus-reactive Th-cells in vitro in H-2d mice. Two of these peptides (4-6 and 17) were able to prime virus-reactive Th-cells in H-2b mice. Nine peptides primed outbred mice in vitro for an antiviral antibody response significantly greater than that seen in animals primed with an irrelevant peptide. These results correlate with, and expand on, our previous observations based on a smaller set of synthetic peptides derived from the E-glycoprotein of Murray Valley encephalitis virus and suggest that synthetic peptides can function as E-glycoprotein Th-cell epitopes. The similarity of results between two distantly related flaviviruses suggests that E-glycoprotein Th-cell epitopes are consistent in location and activity.

OBJECTIVE

The purpose of this study was to determine the relationship between fetal inflammatory and immune responses to oral pathogens and risk for preterm birth.

METHODS

Six hundred and forty umbilical cord blood specimens were prospectively collected. Cord serum levels of C-reactive protein, <em>interleukin</em> (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, Prostaglandin E2, and 8-isoprostane were determined by enzyme-linked immunosorbent assay and categorized as>> median (high) versus < or = median (low). Presence of fetal immunoglobulin M (IgM) antibody against oral pathogens was determined by checkerboard immunoblot assay; detection of>> or = 1 oral pathogen specific antibody was categorized as positive. Preterm birth was defined as spontaneous delivery at (<em>35</em> weeks. Chi-square analysis was used to determine association between cord serum mediator or IgM category and preterm birth. Odds ratios (OR) for preterm birth were calculated, stratified by mediator and IgM category.

RESULTS

Of 640 births, 48 (7.5%) delivered preterm. Preterm birth rates were higher if categorized as high versus low 8-isoprostane or TNF-alpha (23 vs 5%, P < .001 and 10 vs 4%, P < .01, respectively). Preterm birth rates were also higher if categorized as IgM positive versus negative (10.6 vs 5.8%, P = .04). The joint effects of fetal IgM seropositivity, detectable C-reactive protein, or high 8-isoprostane, PGE(2), or TNF-alpha resulted in significantly increased risk for preterm birth (adjusted OR [95% CI]: 6.0 [2.2-16.5], 4.3 [1.6-11.5], 4.1 [1.5-11.6], and 7.6 [2.3-20.8], respectively).

CONCLUSIONS

Fetal exposure to oral pathogens evidenced by an IgM response is associated with preterm birth, and the risk for preterm birth is greatest among fetuses that also demonstrate an inflammatory response.

We have reported that proteasomes are expressed at abnormally high levels in various hematopoietic tumor cells (Kumatori, A., Tanaka, K., Inamura, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawa, T., Shin, S., and Ichihara, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7071-7075). In the present study, we examined changes in the expressions of proteasomes during growth of peripheral T-lymphocytes from healthy adults and differentiation of human leukemic cell lines. Up-regulation of mRNAs encoding multiple proteasome subunits was observed during proliferation of resting T-cells induced by mitogens such as phytohemagglutinin and <em>interleukin</em>-2. In contrast, in vitro terminal differentiation into monocytic, granulocytic, and erythroid cells of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells, by various inducing agents caused rapid and marked down-regulation of proteasomes expression, independently of the cell type, direction of differentiation, or type of signal. The syntheses of proteasome subunits of 21-31 kDa and their associated components of <em>35</em>-110 kDa, measured by [<em>35S</em>]methionine incorporation, were much higher in mitogen-activated T-cells and unstimulated HL-60 cells, which grow rapidly, than in resting and differentiated cells, indicating apparent correlations of the mRNA levels of proteasomes with their translational activities. However, immunochemically, no detectable difference in the cellular contents of proteasomes was found in these cells in induced and uninduced states for proliferation and differentiation, suggesting accelerated turnover of proteasomes in rapidly proliferating cells. Inhibition of proteasome expression by an antisense oligodeoxynucleotide for the largest proteasome subunit, C2, caused partial arrest of cell cycle progression of T-lymphocytes, suggesting that up-regulation of proteasomes is indispensable for proliferation of the cells. We also observed that the nuclear fraction of proteasomes increased in proliferating T-cells and that proteasomes moved rapidly between the nucleus and cytoplasm during differentiation of HL-60 cells.

Studies in humans and in experimental models suggest the involvement of the immune system for efficacy of drug treatment against protozoan parasites. This study tested this hypothesis by using various cytokine and inducible nitric oxide synthase (iNOS) knockout (KO) mice infected with Trypanosoma cruzi and treated with benznidazole. In contrast with the 100% parasitologic cure rate achieved in wild-type animals, benznidazole failed to cure 100%, 42%, <em>35</em>%, and 28% of interferon-gamma, <em>interleukin</em>-12 (protein 40), protein 55-tumor necrosis factor receptor, and iNOS KO mice, respectively. These results suggest that activation of the immune system by the parasite and endogenous interferon-gamma play a major role in the efficacy of benznidazole against infection with T. cruzi.

The function of two alpha-helical regions of mouse <em>interleukin</em>-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu<em>35</em> and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and <em>35</em> had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.

Primary infection with Epstein-Barr virus (EBV) may arise as infectious mononucleosis (IM) in adolescents and young adults. Morphologically, IM-affected lymphoid tissue is characterized by expanded interfollicular areas with formation of atypical lymphoid blasts. It is assumed that morphology and clinical presentation of IM are related to characteristic patterns of cytokine production by EBV-infected and reactive cells. We studied IM tonsils of eight patients and six normal tonsils with a double in situ hybridization procedure using [<em>35S</em>]-labeled RNA probes specific for various cytokines and digoxigenin-labeled probes for the detection of the nuclear EBV encoded RNA transcripts, EBER 1 and 2. All of the IM cases displayed the same distinct cytokine gene expression pattern. When compared with interfollicular areas of normal tonsils, expression of lymphotoxin (LT), tumor necrosis factor-alpha (TNF-alpha), <em>interleukin</em>-6 (IL-6), and IL-1 beta, but not IL-8 or IL-1 alpha was strongly enhanced in interfollicular areas in IM tonsils. LT was expressed predominantly by EBV-infected cells. TNF-alpha transcripts were also present in EBV-infected cells, although in smaller proportions. IL-6 specific signals were only found in few EBV-infected cells. IL-1 alpha-, IL-1 beta-, and IL-8-specific signals were not observed in EBV-infected cells, but were present at high signal intensity in many cells within and around foci of EBV-infected cells (IL-1 beta), next to areas of necrosis (IL-8, IL-1 beta), or in epithelial cells (IL-1 alpha). These data suggest that EBV infection in form of IM results in induction of specific sets of cytokine genes in EBV-infected and in neighboring EBV-negative cells contributing to the characteristic morphology and cellular arrangement of the lesion as well as the clinical presentation.

We examined the ability of human recombinant <em>interleukin</em>-1 beta (hrIL-1 beta) to alter the release of [3H]norepinephrine ([3H]NE) by KCl or electrical field stimulation in longitudinal muscle-myenteric plexus of rat intestine. The cytokine had no immediate effect on either the basal or evoked release of [3H]NE. However, hrIL-1 beta caused a biphasic time-dependent suppression of evoked [3H]NE release that was delayed in onset. IL-1 beta also stimulated the cycloheximide-sensitive uptake of [<em>35S</em>] methionine uptake by the tissue. The initial suppression of [3H]NE release was observed after 30 min and could not be inhibited by cycloheximide. A delayed peak was observed after 120 min and was inhibited by cycloheximide. The effect of IL-1 beta was maximal at 10 ng/ml and could be prevented by a neutralizing anti-IL-1 beta antibody or by preincubating the tissue with an IL-1-receptor antagonist. These results indicate that IL-1 beta suppresses [3H]NE release from rat myenteric plexus by two mechanisms, one of which is independent of protein synthesis and the other of which is mediated by endogenous IL-1.