Background: Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients.

Methods: A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival.

Results: In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6-24.0, P = 0.52) and 22.4% (97.5% CI: 17.2-28.3, P < 0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline.

Conclusions: Tocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline. Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092).

Keywords: COVID-19; Coronavirus; IL-6; Mortality; Phase 2; Pneumonia; Safety; Tocilizumab.

This study describes the identification and characterization of a soluble <em>interleukin</em>-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of <em>35</em>-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.

In the last three decades, numerous reports have shown that patients with chronic pulmonary disease and with heart failure with hypoxemia cleared drugs at a lower rate than healthy volunteers. As a consequence decreased clearance, drug toxicity is frequent in these patients. The reduction in drug clearance is due to a decrease in activity of cytochrome P450 isoforms, partly associated to the hypoxemia. With in vivo animal models, acute moderate hypoxia (PaO2 of around <em>35</em>-50 mm Hg) reduces the clearance of drugs biotransformed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2E1, although hypoxia does not affect the clearance of drugs biotransformed by CYP3A6. Ex vivo and in vitro experiments demonstrate that hypoxia down-regulates CYP1A1, CYP1A2, CYP2B6, CYP2C9 and CYP2C19, decrease preceded by a reduction in activity. On the other hand, acute moderate hypoxia up-regulates CYP3A6. The changes in protein expression are preceded by modifications in the mRNA coding for the proteins. The effect of hypoxia on hepatic cytochrome P450 is carried out by serum mediators, e.g. interferon-gamma, <em>interleukin</em>-1beta, and <em>interleukin</em>-2 are responsible for the decrease in activity and in expression of cytochrome P450 isoforms, and erythropoietin accounts for the increase in CYP3A6. Probably several mechanisms underlie and contribute to the decrease in activity and down-regulation of cytochrome P450 isoforms by hypoxia, e.g. reducing potentiation factors, inducing repressor elements and activating negative regulatory elements. The up-regulation of CYP3A6 implies a PTK- and p42/44MAPK-dependent stabilization/activation, nuclear translocation of HIF-1 and AP-1, binding to CYP3A6 promoter, and transactivation of the gene to induce CYP3A6 expression.

Neurocysticercosis, caused by Taenia solium, is one of the most common causes of seizures worldwide. The symptoms result from granulomatous inflammation associated with dying cyst forms of the parasite. Although the invasive larvae can be killed by immune serum plus complement, immunity to the cyst stage depends on a cellular response. This dichotomous immune response is reminiscent of the extremes of the immune response associated with T helper 1 (Th1) and Th2 cytokine profiles. To characterize the cytokine response in cysticercosis, granulomas were removed from the peritoneal cavity of mice infected with Taenia crassiceps cysts and examined for cytokine message by in situ hybridization using <em>35S</em>-labeled RNA probes. The granulomas were staged based on histologic appearance of the degenerating parasite. Message for gamma interferon (IFN-gamma) was identified by light microscopy in 11 of the 12 granulomas, and <em>interleukin</em>-2 (IL-2) message was identified in 9 of the 12. By laser scanning confocal microscopy, significantly increased IFN-gamma and IL-2 pixel intensity was identified in nearly all of the granulomas from early histologic stages. Message for IL-4 was seen in 6 of the 12 granulomas. Only granulomas with complete destruction of the parasite architecture displayed more than minimal amounts of IL-4 message by light microscopy, and only 2 of 12 granulomas had IL-4 pixel intensity significantly above background. Only minimal amounts of IL-10 message were detected in 4 of 11 granulomas. Thus, early granulomas in cysticercosis are predominantly associated with a Th1 response, whereas later granulomas, in which parasite destruction is complete, have a mixture of Th1 and IL-4. The Th1 response appears to play an important role both in the pathogenesis of disease as well as in the clearing of the parasites, with IL-4 involved in downregulation of the initial response.

Oxidative stress and inflammation have not been well-characterized in extreme pediatric obesity. We compared levels of circulating oxidized low-density lipoprotein (oxLDL), C-reactive protein (CRP), and <em>interleukin</em>-6 (IL-6) in extremely obese (EO) children to normal weight (NW) and overweight/obese (OW/OB) children. OxLDL, CRP, IL-6, BMI, blood pressure, and fasting glucose, insulin, and lipids were obtained in 225 children and adolescents (age 13.5 ± 2.5 years; boys 55%). Participants were classified into three groups based on gender- and age-specific BMI percentile: NW (<85th, n = 127), OW/OB (85th- <1.2 times the 95th percentile, n = 64) and EO (≥1.2 times the 95th percentile or BMI ≥<em>35</em> kg/m(2), n = 34). Measures were compared across groups using analysis of covariance, adjusted for gender, age, and race. Blood pressure, insulin, and lipids worsened across BMI groups (all P < 0.0001). OxLDL (NW: 40.8 ± 9.0 U/l, OW/OB: 45.7 ± 12.1 U/l, EO: 63.5 ± 13.8 U/l) and CRP (NW: 0.5 ± 1.0 mg/l, OW/OB: 1.4 ± 2.9 mg/l, EO: 5.6 ± 4.9 mg/l) increased significantly across BMI groups (all groups differed with P < 0.01). IL-6 was significantly higher in EO (2.0 ± 0.9 pg/ml) compared to OW/OB (1.3 ± 1.2 pg/ml, P < 0.001) and NW (1.1 ± 1.0 pg/ml, P < 0.0001) but was not different between NW and OW/OB. Extreme pediatric obesity, compared to milder forms of adiposity and NW, is associated with higher levels of oxidative stress and inflammation, suggesting that markers of early cardiovascular disease and type 2 diabetes mellitus are already present in this young population.

OBJECTIVE

Antigen expression intensity is becoming important for decision-making in relation to monoclonal antibody therapy. By quantifying CD20, CD22 and CD52 expression on chronic lymphocytic leukemia and normal (control) B cells, over time. The effect of Interleukin-4 therapy on CD20 antigen intensity on B-CLL cells in vivo was also determined.

METHODS

Lymphocytes were purified at weeks 0, 4 and 8 from five B-CLL patients, five healthy volunteers and seven B-CLL patients receiving IL-4 therapy. The number of antigen receptor sites was calculated in molecules of equivalent soluble fluorochrome using flow cytometry.

RESULTS

The mean number of CD20 receptors at baseline was significantly lower on B-CLL cells compared to normal B cells (8160 vs 87 046; P<0.0001). Similar results were obtained for CD22 (8630 vs 27 647; P<0.01), but not for CD52 (371 303 vs 409 484; P = 0.54). When soluble fluorochrome values at weeks 4 and 8 were analysed as change in per cent from baseline (delta%), there was <10 delta% variability in CD20 expression on control B cells, but considerable variability (22.5-67.5 delta%) on B-CLL cells. Expression of CD22 in CLL and control B cells varied by <15 delta%. CD52 on CLL B cells showed slightly greater variability (+/-35 delta%) than that of CD22 (+/-15delta%), but less than that of CD20. IL-4 therapy did not consistently increase the CD20 expression on B-CLL cells in vivo.

CONCLUSIONS

Our data confirm differences in intensity between different target antigens on B-CLL cells, and draws attention to the fact that a substantial variability may occur over time, which may influence clinical decision-making. Caution must be taken when interpreting in vitro results on cytokine-mediated receptor intensity up-regulation.

OBJECTIVE

Our purpose was to (1) determine the value of amniotic fluid interleukin-6 determination in the detection of microbial invasion of the amniotic cavity and (2) compare interleukin-6 to other rapid tests in the evaluation of preterm labor.

METHODS

Amniotic fluid interleukin-6 was determined quantitatively by enzyme-linked immunosorbent assay in 91 amniotic fluid specimens obtained by amniocentesis in 89 patients with preterm labor. Amniotic fluid cultures for aerobes, anaerobes, and mycoplasma species were performed. Receiver-operator characteristic curve analysis, logistic regression analysis, and Cox's proportional-hazards model were used to explore the relationship between several explanatory and outcome variables. Diagnostic index values of interleukin-6, glucose level, Gram stain, leukocyte esterase, and limulus amebocyte lysate assay for prediction of a positive amniotic fluid culture, preterm delivery, clinical infection, and neonatal sepsis were calculated.

RESULTS

The prevalence of positive amniotic fluid cultures was 13% (12/89). The median interleukin-6 concentration in patients with positive cultures was 241.8 ng/ml, in contrast to 0.291 ng/ml in patients with negative cultures (p. < 0.005). Sensitivity and specificity of an interleukin-6 level>> or = 6.17 ng/ml was 75% and 79%, in contrast to that of glucose, < or = 12 mg/dl (83% and 86%) for a positive amniotic fluid culture and sensitivity (p = 0.26, z test). All patients with an interleukin-6 level>> 6.17 ng/ml were delivered preterm, in contrast to 85.2% of patients with a glucose level < or = 12 mg/dl. When all rapid tests and clinical parameters were considered simultaneously in the logistic model, only interleukin-6 maintained a significant relationship to preterm birth (odds ratio 35, p = 0.003). Cox's proportional analysis demonstrated a strong relationship between interleukin-6 and the amniocentesis-to-delivery interval after clinical variables were controlled for (hazard ratio 3.01, p < 0.00001).

CONCLUSIONS

Amniotic fluid interleukin-6 determination may be a useful adjunct to our armamentarium of rapid tests to exclude infection and predict delivery in patients with preterm labor and intact membranes.

Coxsackievirus and adenovirus receptor (CAR), alphav integrins, and heparan sulfate glycosaminoglycans (HSGs) are the tropism determinants of adenoviral (Ad) vectors in vivo. For the development of a targeted Ad vector, its broad tropism needs to be blocked (or reduced). We have previously developed Ad vectors with ablation of CAR, alphav integrin, and HSG binding by mutation of the FG loop in the fiber knob (deletion of T489, A490, Y491, and T492 of the fiber protein), deletion of the RGD motif of the penton base, and substitution of the fiber shaft domain for that derived from Ad type <em>35</em>, respectively, and have shown that this triple-mutant Ad vector [Ad/deltaF(FG)deltaP-S<em>35</em>-L2] exhibits significantly lower transduction in mouse liver compared with the conventional Ad vector [Koizumi, N., Mizuguchi, H., Sakurai, F., Yamaguchi, T., Watanabe, Y., and Hayakawa, T. (2003). J. Virol. 77, 13062-13072]. In the present study, we optimized the fiber knob mutation for further reduced in vivo transduction and examined toxicity of the modified Ad vectors. Ad/deltaF(AB)deltaPS<em>35</em>- L2, a triple-mutant Ad vector containing a mutation of the AB loop in the fiber knob (R412S, A415G, E416G, and K417G), mediated approximately 15,000- and 500-fold lower mouse liver transduction by intravenous and intraperitoneal administration, respectively, than the conventional Ad vector, and mediated 10- fold lower mouse liver transduction than did Ad/deltaF(FG)deltaP-S<em>35</em>-L2. Ad/deltaF(AB)deltaP-S<em>35</em>-L2 also exhibited lower transduction of other organs compared with Ad/deltaF(FG)deltaP-S<em>35</em>-L2 and the conventional Ad vector. Levels of both liver serum enzymes (aspartate transferase [AST] and alanine transferase (ALT)] and <em>interleukin</em> (IL)-6 in mouse serum after intravenous administration of Ad/deltaF(AB)deltaP-S<em>35</em>-L2 were similar to those in the nontreatment mouse serum, whereas the conventional Ad vector led to high levels of AST, ALT, and IL-6. We therefore succeeded in further improving the mutant Ad vector, abolishing both viral natural tropism and toxicity. This new Ad vector appears to be a fundamental vector for targeted gene delivery.

OBJECTIVE

To study a possible correlate of protection in mother-to-infant transmission of HIV infection. In particular, to determine whether lack of HIV-specific T-helper (TH) function as indicated by HIV and non-HIV antigen-stimulated interleukin (IL)-2 production of mother and/or newborn peripheral blood leukocytes (PBL) is associated with mother-to-infant transmission of HIV.

METHODS

PBL from 21 HIV-seropositive pregnant women and 23 cord blood leukocytes (CBL) from their offspring were studied for in vitro TH function by IL-2 production in response to HIV and non-HIV antigens. Polymerase chain reaction (PCR) and viral culture assays were performed to determine HIV infection of the infants.

RESULTS

PBL from 10 out of 21 (48%) mothers and from eight out of 23 (35%) CBL samples responded to two or more out of five synthetic gp 160 envelope (env) peptides. Three of the 23 (13%) offspring were shown to be HIV-infected by PCR and/or viral culture on follow-up. All three infected infants were from a subset whose CBL did not exhibit env-specific TH immunity.

CONCLUSIONS

Our results demonstrate that fetal T cells can be primed to HIV env determinants in utero, suggest that HIV-specific TH immunity may be protective in newborns, and provide a possible means for identifying newborns who are at risk for HIV infection.

Glycogen synthase kinase 3beta (GSK-3beta) is a serine/threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. Dysregulation of GSK-3beta has been implicated in the pathogenesis of several diseases including sepsis. Here we investigate the effects of 2 chemically distinct inhibitors of GSK-3beta, TDZD-8 and SB216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. Male Wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to <em>35</em> mmHg for 90 min) and subsequently resuscitated with shed blood for 4 h. Hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. Treatment of rats with either TDZD-8 (1 mg/kg, i.v.) or SB216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. In addition, TDZD-8, but not SB216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine <em>interleukin</em> 6 and also of the anti-inflammatory cytokine <em>interleukin</em> 10. Neither of the GSK-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. Thus, we propose that inhibition of GSK-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock.

15N NOE, T1, and T2 measurements have been carried out on uniformly 15N-labeled human <em>interleukin</em>-4. Analysis of the results in terms of order parameters (S2) shows that although the helical core of this four-helix-bundle protein exists as a well-defined structure with limited conformational flexibility (S2 congruent to 0.9), other regions of the molecule experience substantial fluctuations in the conformation of the main chain (S2 = 0.3-0.8). These regions include both the N- and C-termini and two of the loops joining the helices. The majority of these internal motions are fast compared with the overall rotational correlation time (tau R = 7.6 ns at <em>35</em> degrees C) and are localized in regions that are relatively ill-defined in the NMR structures previously determined for this protein [Smith, L. J., Redfield, C., Boyd, J., Lawrence, G. M. P., Edwards, R. G., Smith, R. A. G., & Dobson, C. M. (1992) J. Mol. Biol. 224, 899-904]. Other motions are on a slower time scale and appear to be associated with two of the three disulfide bonds and the beta-sheet region in the protein. The dynamic properties of <em>interleukin</em>-4 in solution have been compared with features of the X-ray structures of other four-helix-bundle proteins. The results suggest that the dynamic properties observed here may be general for this class of proteins and may be significant for the interpretation of both their structural and functional properties.

<em>Interleukin</em>-2 (IL-2) targets cells bearing IL-2 receptors and induces different degrees of lymphocytosis. This study retrospectively evaluated whether lymphocytosis, in addition to clinical characteristics at baseline and to tumor objective response, may predict overall survival in metastatic renal cell carcinoma patients who received IL-2 subcutaneously (s.c.). Overall survival, clinical characteristics, tumor response, and total lymphocyte count at baseline and during the first treatment cycle of 266 advanced renal cell cancer patients, treated with 1 of 4 different first-line s.c. IL-2-based protocols, were studied using the Cox multivariate analysis. Median IL-2 cumulative dose and length of treatment (+/-SD) were 232 +/- 282 x 10(6)/m(2) in 7 +/- 5.9 weeks, respectively. Median overall survival (os) was 13.1 months (range 0.7-86.9+) in all. Tumor outcome consisted of: 9 CR (3%) (os = NR); <em>35</em> PR (13%) (os = 19.7 months.); 117 SD (44%) (os = 15.1 months); 105 PD (39%) (os = 6.4 months). Median lymphocyte counts were 1400/mm(3) at baseline (25th-75th, 900-1900/mm(3)) and 3600/mm(3) as a maximum value (25th-75th, 2600-4800/mm(3)). Death risk significantly decreased by 11% for each 1,000 lymphocytes/mm(3) (RR 0.89; 95% CI 0.82-0.97), after correcting for clinical characteristics (PS ECOG 0 versus>> or =1, time from primary diagnosis>> or =2 years versus <2 years, number of metastatic sites 1 versus >1) and tumor response (CR, PR). A two-step bootstrapping procedure confirmed such predictive performance. Lymphocyte count monitoring represents a biomarker of the host response to subcutaneous IL-2 treatment useful for multimodal clinical assessment, as it predicts overall survival in advanced cancer patients independently from tumor response and from main clinical characteristics.

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as <em>interleukin</em>-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [<em>35S</em>]methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [<em>35S</em>]methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-alpha and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.

BACKGROUND

The principal components of the interleukin-1 (IL-1) family are two secreted factors (IL-1alpha and IL-1beta), two transmembrane receptors (IL-1RI [biologically active] and IL-1RII [inert receptor]), and a natural antagonist receptor of IL-1 function (IL-1Ra). Changes in the expression pattern of these IL-1 members have been reported to be related to disease progression. The objective of the current study was to evaluate these changes in prostatic tissue by means of immunohistochemistry and Western blot analysis.

METHODS

Immunohistochemical and Western blot analyses were performed in 20 normal samples, 35 samples of benign prostatic hyperplasia (BPH) and 27 samples from patients with prostate carcinoma (PC).

RESULTS

In normal prostate samples, immunoreactions to IL-1beta and IL-1RI were positive, whereas there were no immunoreactions observed to IL-1alpha, IL-1RII, or IL-1Ra. In BPH, in addition to immunoreactions to IL-1beta and IL-1RI, immunoreactions to IL-1alpha, IL-1RII, and IL-1Ra were observed in many samples. In samples of PC with low Gleason grade, most tumors had positive immunoreactions to IL-1alpha and IL-1RI. In samples of PC with high Gleason grade, immunoreactions were seen only to IL-1alpha, IL-1RI, and IL-1RII.

CONCLUSIONS

The current results suggested that high expression levels of IL-1alpha and IL1-RI in epithelial cells in BPH and PC samples were involved in cell proliferation and that the loss of immunoexpression of IL-1beta and IL-1Ra was a characteristic feature of PC compared with normal prostate samples and BPH. Because this loss is progressive up to a complete absence of immunoexpression in PC of high Gleason grade, the evaluation of IL-1beta and IL-1Ra in PC may be significant in assessing for malignancy.

We investigated whether gamma interferon (IFN-gamma; a Th1 cytokine), tumor necrosis factor alpha (TNF-alpha), and <em>interleukin</em>-4 (IL-4; a Th2 cytokine) modulate nitric oxide (NO) production in vivo during blood stage infection with Plasmodium chabaudi AS. Treatment of resistant C57BL/6 mice, which resolve infection with P. chabaudi AS and produce increased levels of IFN-gamma, TNF-alpha, and NO early during infection, with anti-IFN- gamma plus anti-TNF-alpha monoclonal antibodies (MAbs) resulted in a reduction of both splenic inducible NO synthase mRNA and serum NO3- levels by 50 and 100%, respectively. Treatment with the anti-TNF-alpha MAb alone reduced only serum NO3- levels by <em>35</em>%, and treatment with the anti-IFN-gamma MAb alone had no effect on NO production by these mice during infection. Susceptible A/J mice, which succumb to infection with P. chabaudi AS and produce increased levels of IL-4 but low levels of IFN-gamma, TNF-alpha, and NO early during infection, were treated with an anti-IL-4 MAb. The latter treatment had no effect on NO production by this mouse strain during infection. In addition, our results also demonstrate that treatment of resistant C57BL/6 mice with anti-IFN-gamma plus anti-TNF-alpha MAbs affects, in addition to NO production, other traits of resistance to P. chabaudi AS malaria such as the peak level of parasitemia and the development of splenomegaly. Furthermore, the change in spleen weight was shown to be an IFN-gamma-independent effect of TNF-alpha. Treatment of susceptible A/J mice during infection with an anti IL-4 MAb had no effect on these markers of resistance. Thus, these results demonstrate that TNF-alpha and IFN-gamma are critical in the regulation of NO production and other traits of resistance during P. chabaudi AS malaria in C57BL/6 mice. These data also indicate that treatment with an anti-IL-4 antibody alone is not able to induce NO production or confer resistance to A/J mice against P. chabaudi AS malaria.

Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, <em>interleukin</em> 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[<em>35S</em>]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

Donor T cells activated by recipient alloantigens cause graft-versus-host disease (GVHD) after hematopoietic cell transplantation. Activated T cells express CD25, among other components of the <em>interleukin</em>-2 receptor. We conducted a phase I/II study to determine whether administration of CD25-specific antibody conjugated to ricin toxin A could reduce the risk of grade III or IV GVHD after marrow transplantation from HLA-matched unrelated donors. All patients received methotrexate and cyclosporine after the transplantation. The immunotoxin was given to 36 patients for 4 consecutive days beginning approximately 36 hours after the marrow infusion was completed. Fourteen (40%) of the <em>35</em> patients who could be evaluated developed grade III or IV GVHD. In a contemporaneous population of 121 patients who received marrow from HLA-matched unrelated donors and were given methotrexate and cyclosporine without the immunotoxin, the incidence of grades III and IV GVHD was 24%. Cyclosporine blocked the induction of CD25 expression on alloactivated T cells in vitro but had no detectable effect on CD25 expression by T-regulatory cells. Taken together, these results are consistent with the hypothesis that cyclosporine protected alloactivated donor T cells from the effects of the immunotoxin, whereas the CD25+ T-regulatory cells remained susceptible, causing an unexpected exacerbation of acute GVHD.

OBJECTIVE

WX-G250 is a chimeric monoclonal antibody that binds to carbonic anhydrase IX(G250/MN), which is present on greater than 95% of RCCs of the clear cell subtype. The suggested working mechanism of WX-G250 is by ADCC. Because the number of activated ADCC effector cells can be increased by a low dose interleukin-2 pulsing schedule, a multicenter study was initiated to investigate whether WX-G250 combined with LD-IL-2 could lead to an improved clinical outcome in patients with progressive RCC.

METHODS

A total of 35 patients with progressive clear cell RCC received weekly infusions of WX-G250 for 11 weeks combined with a daily LD-IL-2 regimen. Patients were monitored longitudinally for ADCC capacity. Radiological assessment of metastatic lesions was performed at week 16 and regularly until disease progression.

RESULTS

A durable clinical benefit was achieved in 8 of 35 patients (23%), including 3 with a partial response and 5 with stabilization at 24 weeks or greater. Mean survival was 22 months. In general treatment was well tolerated with little toxicity. The number of effector cells increased during treatment but lytic capacity per cell did not increase. ADCC and clinical outcome did not appear to correlate.

CONCLUSIONS

WX-G250 combined with LD-IL-2 in patients with metastatic RCC is safe and well tolerated. With a substantial clinical benefit and a median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry combination therapy showed increased overall survival compared to WX-G250 monotherapy. Survival was at least similar to that of currently used cytokine regimens but with a favorable toxicity profile.

This study investigates the ability of P388D1 macrophages to synthesize and secrete substance P (SP). Using a monoclonal anti-SP antibody (termed MASP-1) coupled to Sepharose, it was possible to immunoaffinity purify from culture supernates a peptide that was antigenically related to SP. P388D1 macrophage cultured in the presence of <em>35S</em>-methionine secreted into culture supernates a labelled peptide which could be recognized by MASP-1. Affinity-purified, P388D1-derived SP was shown to be chemically similar to synthetic SP using gel-filtration chromatography and reverse-phase HPLC. In addition, an RIA using a polyclonal, monospecific antibody was used to quantify the amount of secreted SP in cultured supernates. P388D1 macrophages secreted 222 pg SP per 10(8) cells, whereas SP secretion by control thymocyte cultures was not detectable. The functionality of the P388D1-derived SP was also investigated. Since exogenously added SP can increase secretion of an <em>interleukin</em>-1 (IL-1)-like activity in these cells, we questioned whether an anti-SP antibody could remove P388D1-secreted SP from the culture, and in turn reduce cytokine production. By culturing varying dilutions of MASP-1 with P388D1 cells, it was possible to decrease cytokine production by P388D1 cells compared to cultures containing no antibody or with normal mouse immunoglobulin G (IgG). Taken together, these studies demonstrate that macrophage-derived SP may function in an autocrine or paracrine fashion to modulate macrophage function.

We analyzed the long-term clinical course of 71 patients with RNA-positive hepatitis C virus (HCV) infection after liver transplantation. Patients with reinfection after transplantation for HCV-related liver disease, or de novo infection at transplantation were followed for up to 12 years. Cumulative survival for patients with HCV infection at 2, 5, and 10 years after transplantation was 67%, 62%, and 62%, respectively. It was not significantly different from that in patients transplanted for other nonmalignant diseases without HCV infection. The main factor determining long-term survival was the presence or absence of hepatocellular carcinoma (HCC) at transplantation. The 5-year survival rate for HCV patients with or without HCC was <em>35</em>% versus 73%, respectively (P < .05). No deaths because of viral hepatitis of the graft were observed. Deaths in the first year after transplantation were caused by infectious complications, cardiovascular problems, or rejection; deaths after more than 12 months were exclusively because of recurrence of HCC. Biochemical and histological evidence of hepatitis was found in the majority of the patients, only 16% had normal alanine aminotransferase (ALT) values throughout. Twenty-two percent of patients complained of symptoms, with hepatitis C being the cause in 82% of these. Two patients lost their HCV-RNA for prolonged, ongoing periods of time. The severity of the posttransplantation hepatitis was unrelated to age, sex, severity of liver disease before transplantation, cold ischemic time of the graft, duration of the operation, transfusions, the number of rejection episodes, or the long-term immunosuppressive regime. Only initial short-term therapy with <em>interleukin</em> 2 (IL2) receptor antibodies adversely influenced inflammatory activity. Viral genotype did not influence the course of the graft hepatitis in our series. Histology showed inflammation in 88% of the biopsies and signs of fibrosis in 24%. Mean ALT values correlated with inflammation but not with fibrosis in the biopsies. Porto-portal bridging was observed in six patients, one patient developed cirrhosis within 2 years after orthotopic liver transplantation (OLT). We conclude that chronic hepatitis develops in the majority of patients with HCV infection after liver transplantation. Carrier states without significant laboratory abnormalities are observed in approximately 16%, biochemical abnormalities without symptoms are seen in 60%, and symptomatic disease develops in a quarter of the patients. The disease course closely resembles that seen in nontransplanted hepatitis C patients. It is generally mild but little over 10% of patients develop signs of fibrosis of the graft during the first decade.

Studies indicate that, whereas immune functions in males are depressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se is responsible for producing the immune depression. To study this, female C3H/HeN mice (n = 7 animals/group) were pretreated with 5-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock (blood pressure <em>35</em> +/- 5 mmHg for 90 min) followed by fluid resuscitation or sham operation. Nontreated males underwent either trauma hemorrhage or sham operation. Twenty-four hours thereafter, splenocyte immune functions as well as plasma DHT, estradiol, and corticosterone levels were measured. DHT-pretreated females had significantly (P < 0.05) increased DHT levels, comparable to those seen in males. Conversely, estradiol levels in such females were similar to control males. Splenocyte proliferation as well as <em>interleukin</em>-2 and <em>interleukin</em>-3 release were not depressed in vehicle-treated females, whereas it was in DHT-treated females after trauma hemorrhage, comparable to hemorrhaged males. Thus high testosterone and/or low estradiol levels appear to be responsible for producing splenocyte immune depression in males after trauma hemorrhage. Agents that block testosterone receptors or increase estradiol levels may therefore be helpful in improving depressed immune functions in male trauma patients.

Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma-free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with <em>interleukin</em>-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( < or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (<em>35</em> to 60 days) and extended long-term culture initiating cell (ELTC-IC) period (>> 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P < .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P < .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro.

Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine <em>interleukin</em> (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an effective systemic antitumoral immunity in the host and prolonged the median survival time of the treated animals from 22 to <em>35</em> days. The antitumoral immunity declined gradually, which led to tumor recurrence over time. A recombinant adenovirus expressing the mIL-12 gene was constructed and tested in the MCA-26 tumor model. Intratumoral administration of this cytokine vector alone increased significantly survival time of the animals with 25% of the treated animals still living over 70 days. These data indicate that local expression of IL-12 may also be an attractive treatment strategy for metastatic colon carcinoma.

We investigated the expression of Th1- and Th2-associated chemokine receptors on peripheral blood lymphocytes at diagnosis and in the first phase of type 1 diabetes. Peripheral blood mononuclear cells (PBMCs) of 25 patients with newly diagnosed type 1 diabetes, 10 patients with longstanding type 1 diabetes, and <em>35</em> healthy control subjects were examined for expression of the chemokine receptors CXCR4 (naive T-cells), CCR5 and CXCR3 (Th1 associated), and CCR3 and CCR4 (Th2 associated) on CD3+ lymphocytes. Furthermore, we analyzed chemokine serum levels (monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, and RANTES [regulated on activation, normal T-cell expressed and secreted]) and phytohemagglutinin (PHA)-stimulated cytokine secretion of Th1- (gamma-interferon [IFN-gamma] and tumor necrosis factor-alpha [TNF-alpha]) and Th2 (<em>interleukin</em> [IL]-4 and -10)-associated cytokines by PBMC. The patients with newly diagnosed type 1 diabetes were followed for these parameters at 6-12 months after diagnosis. The PBMCs of patients with newly diagnosed but not with longstanding type 1 diabetes showed reduced expression of the Th1-associated chemokine receptors CCR5 (P < 0.001 vs. control subjects) and CXCR3 (P < 0.002 vs. control subjects). This reduction correlated with reduced IFN-gamma and TNF-alpha production of PBMCs after PHA stimulation and reversed 6-12 months after diagnosis to normal levels. CCR4 cells were reduced in both newly diagnosed and longstanding type 1 diabetic patients, which correlated to reduced PHA-stimulated IL-4 production. MIP-1alpha and MIP-1beta levels were considerably elevated in a subgroup of patients with newly diagnosed diabetes. We assume that Th1-associated peripheral T-cells are reduced in a narrow time window at the time of diagnosis of diabetes, possibly due to extravasation in the inflamed pancreas. Thus, chemokine receptor expression of peripheral blood lymphocytes may be a useful surrogate marker for the immune activity of type 1 diabetes (e.g., in intervention trials).