Oncolytic virus in gliomas: a review of human clinical investigations.

Gliomas remain one of the more frustrating targets for oncologic therapy. Glioma resistance to conventional therapeutics is a product of their immune-privileged milieu behind the blood-brain barrier, in addition to their suppressive effect on the immune response itself. Taking the lead from the growing success of immunotherapy for systemic cancers, such as lung cancer and melanoma, immunotherapeutics has emerged as a major player in the potential treatment of gliomas, with oncolytic viruses in particular showing significant promise as evidenced by the recent Breakthrough and Fast Tract Designations for PVSRIPO and DNX2401. This review serves as a useful and updated compendium of the completed human clinical investigations for several oncolytic viruses in the treatment of gliomas.

Development of the antinuclear and anticytoplasmic antibody consensus panel by the Association of Medical Laboratory Immunologists.

The Association of Medical Laboratory Immunologists (AMLI) have developed a panel of antinuclear and anticytoplasmic antibody consensus sera that can be useful for enzyme immunoassay (EIA), Ouchterlony, and immunofluorescence assay methods. It was developed to assist in the evaluation of newly available EIA methods for the detection of autoantibodies. The panel of sera was evaluated in several clinical laboratories and a large number of laboratories owned by manufacturers of clinical autoantibody testing kits. The majority of sera performed well for the EIAs in both the clinical laboratories and the manufacturers' laboratories, but some samples had discrepant results. A major source of discrepancy is the current inability of the EIA results to be directly compared in a quantitative way as no standardization exists. The evaluation demonstrated lower sensitivity of detection by the Ouchterlony method. The limited evaluation of the sera with immunoblotting and Western blotting did not show good agreement with other methods. Further work must be done to standardize blotting methods prior to their use in routine clinical testing. The sera are now available to vendors and clinical laboratories for use in the detection of SS-A, SS-B, Sm, U1-RNP, Scl-70, Jo-1, double-stranded DNA, and centromere antibodies. The availability of the consensus sera will help evaluate and improve the EIA methods currently being used.

ThinPrep Pap Test: performance and biopsy follow-up in a university hospital.

BACKGROUND: The ThinPrep Pap Test (TP), a liquid-based cervical cytology preparation, was approved for use in the U.S. in 1996. The purpose of this study was to compare TP performance and biopsy follow-up studies with a similar population of high risk patients sampled by conventional Papanicolaou (Pap) smear (CS). METHODS: Diagnostic and specimen adequacy interpretations for 2727 TP direct-to-vial Pap tests from a high risk university hospital practice were compared with 5000 CS preparations from the same physicians taken 1 year previously. Biopsy follow-up studies for the categories of squamous intraepithelial lesion (SIL), carcinoma, and atypical squamous cells of undetermined significance (ASCUS) for each time period and technique were contrasted. RESULTS: The SIL/carcinoma detection rate increased from 7.7% to 10.5% (P < 0.01) and the ASCUS rate decreased from 12.5% to 6.9% (P < 0.01); the percentage of satisfactory but limited specimens decreased from 19.4% to 10.5% (P < 0.01). Low grade SIL cases increased by 57% (P < 0.01) whereas the 26% increase in high grade SIL cases was not statistically significant. Greater than 90% of ungraded SIL, high grade SIL, and carcinoma cases had abnormal biopsies by both the TP and CS methods. The number of biopsy-confirmed high grade dysplasias and carcinomas was similar in the two groups. A low grade SIL detected by TP was less likely to have an abnormal biopsy (70% vs. 85% for CS). Nevertheless, the 57% increase in low grade SIL diagnoses by TP resulted in more TP patients with dysplastic biopsy diagnoses. Follow-up studies for ASCUS cases diagnosed by either TP or CS were similar, and 21-24% of patients eventually were found to have dysplasia. CONCLUSIONS: The TP technique appears to lead to the increased detection of low grade SIL lesions, decreased satisfactory but limited samples, and fewer equivocal specimens. No increase in biopsy-confirmed high grade dysplasias and carcinomas was found. Follow-up studies for the ASCUS category were nearly identical to those for CS.

Sensitivity and specificity of plasma and urine complement split products as indicators of lupus disease activity.

OBJECTIVE: To determine if measurement of serum complement split products (C4d, Bb, C5b-9) is better than conventional C3 and C4 measurements in distinguishing patients with varying degrees of lupus disease activity, and to determine if the presence of C3d in urine is helpful in distinguishing lupus patients with from those without early lupus nephritis. METHODS: Lupus disease activity was prospectively determined at 3 consecutive visits an average of 4 months apart, using the Systemic Lupus Activity Measure (SLAM), the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and physician global assessment (PGA). Blood samples were evaluated for the presence of C4d, Bb, and C5b-9 by quantitative microassay plate enzyme immunoassay at each patient visit. We characterized urinary excretion of C3 fragments (with attention to C3d) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. RESULTS: Thirty-one SLE patients were enrolled in the study. The mean SLAM score and the mean SLEDAI score each correlated well with the PGA at all 3 visits. A SLAM score of 6 and a SLEDAI score of 4 had the best overall sensitivity and specificity for predicting moderate-to-severe disease activity by PGA (100% and 73%, respectively, for the SLAM and 86% and 94%, respectively, for the SLEDAI). Serum C4d and Bb were more sensitive indicators of current moderate-to-severe lupus disease activity at all 3 visits than were serum C5b-9, C3, and C4. C3 and C4 were more specific indicators of moderate-to-severe disease activity. Serum C4d and Bb were more sensitive at predicting moderate-to-severe disease activity at subsequent visits than were C5b-9, C3, and C4. Urine C3d was better than C3, plasma C4d, Bb, C5b-9 and anti-double-stranded DNA antibody in distinguishing patients with from those without acute lupus nephritis (P = 0.02). CONCLUSION: C4d and Bb are sensitive indicators of moderate-to-severe lupus disease activity and may be most helpful in situations where conventional measurements are not, such as in lupus patients whose C3 and C4 levels remain normal despite evidence of clinical disease activity. It appears from this study that detection of urine C3d may be a simple way of measuring complement activation in the setting of lupus renal disease. The availability of instruments for clinical disease activity measurement such as the SLAM and the SLEDAI may enable more consistent definition of lupus disease activity and may thus provide a means for better examining the role of complement activation products in predicting lupus disease activity in larger patient populations.

Pregnancy outcome and anti-cardiolipin antibody in women with systemic lupus erythematosus.

Pregnancies in women with systemic lupus erythematosus are recognized to result in excessive fetal morbidity and mortality. Maternal autoantibody status may explain some of these problems. Anti-cardiolipin antibody has been associated with recurrent pregnancy losses in some women with lupus, but the risk of these losses has not been defined. At the University of Pittsburgh between January 1, 1979, and December 31, 1989, an unmatched case-control study design was used to determine whether patients with lupus and anti-cardiolipin antibody (81 cases) were at increased risk for adverse pregnancy outcomes in comparison with lupus patients without the antibody (174 controls). Cases had 98 of 192 (51%) pregnancies with an adverse outcome, while controls had 212 of 494 (43%). The odds ratio for having any adverse pregnancy outcome was 1.40 (95% confidence interval (CI) 0.98-1.98). When pregnancies were classified according to specific adverse outcome types, the frequency of late miscarriages (14-20 weeks gestation) in cases and controls was 8% and 3%, respectively. The odds ratio for late miscarriage was 2.94 (95% CI 1.31-6.60). When pregnancies were stratified by birth number and by occurrence of pregnancy before or after diagnosis, the increased frequency of late miscarriages in cases was noted only in the first pregnancy when the pregnancy occurred before recognized disease. Preterm births (before 38 weeks gestation) were increased in cases compared with controls in pregnancies that occurred after diagnosis for second and third pregnancies. If a case had one previous adverse outcome, the odds ratio for another adverse outcome was 3.00 (95% CI 1.62-5.57). If a case had two previous adverse outcomes, the odds ratio for a third adverse pregnancy outcome was 4.14 (95% CI 1.62-10.58). Thus, a previous adverse pregnancy outcome was the most important risk factor for an adverse outcome in a subsequent pregnancy.

Complement C3 fragments in urine: detection in systemic lupus erythematosus patients by western blotting.

We characterized urinary excretion of C3 fragments among patients with systemic lupus erythematosus (SLE) as a possible indicator of renal involvement. 28 patients, representing a broad range of disease activity were admitted to our study. Urinary proteins were separated on 4-20% gradient SDS-PAGE gels, under reducing conditions, and transblotted to nitrocellulose. Western blots were developed with a polyvalent goat-anti-human C3d antiserum, and an alkaline phosphatase-conjugated rabbit anti-goat IgG. Three patterns were obtained: 1) no bands detected; 2) bands suggesting the presence of intact C3; and 3) samples with additional low molecular (< 4 x 10(4)) bands. The 12 patients with no C3 bands had minimal disease activity (e.g. fatigue, arthralgia, arthritis, rash, oral ulcers). The seven patients with intact C3 patterns also had minimally active disease. Their primary clinical findings included fatigue, pleurisy, renal disease which had been treated, hemolytic anemia, and arthritis. Patients with low molecular weight C3 fragments in their urine formed two sub-sets, based upon their presenting features. The first group had severe disease and contained all patients with active lupus nephritis (n = 4), while the second consisted of non-renal patients with primary clinical findings of moderate disease activity (e.g. thrombocytopenia, pneumonitis, arthritis). Our results suggest urinary excretion of low molecular weight C3 fragments correlates with active renal disease, but is a variable finding among SLE patients with non-renal manifestations of disease activity.

Previous pregnancy outcome is an important determinant of subsequent pregnancy outcome in women with systemic lupus erythematosus.

Women with systemic lupus erythematosus (SLE) have increased adverse pregnancy outcomes. The reasons for these problems include maternal disease, clinical or serologic activity, medication use, and residual organ impairment from prior disease flares. In retrospective studies, pregnancy data are often treated cross-sectionally, with births rather than mothers as the unit of analysis. Multiple pregnancies from the same mother may be highly correlated with each other. In an unmatched retrospective study, the first two pregnancy outcomes in lupus patients with anticardiolipin antibody (anti-CL IgG or IgM isotype) (cases N = 47) and without anticardiolipin antibody (controls, N = 125) were assessed according to birth order. A good outcome was defined as a full-term (> 38 weeks) live birth without neonatal complications. All other pregnancy outcomes were considered adverse outcomes. Therapeutic abortions and ectopic or molar pregnancies were excluded. Both cases and controls with an adverse outcome in their first pregnancy had at least a 50% chance of another adverse outcome in their second pregnancy. Cases with a late miscarriage (fetal loss at 14 to 20 weeks' gestation) in their first pregnancy had the highest risk, 80%, of an adverse outcome in their second pregnancy. Both previous pregnancy loss and anti-CL antibody status should be considered in the analysis of pregnancy outcomes in women with SLE.

Elevated IgG anti-histone antibodies in a subgroup of medicated schizophrenic patients.

A total of 57 schizophrenic patients (of which 17 were first-episode, neuroleptic naive) and 76 healthy controls were screened for anti-histone IgG antibodies using an enzyme immunoassay (ELISA). All patients had significantly higher anti-histone antibody titers than controls (t = 3.1, p less than 0.003). Previously medicated patients had significantly higher titers than neuroleptic-naive first episode patients (t = 2.87, p less than 0.006). This study suggests that neuroleptic medications are associated with anti-histone antibodies.

Alterations in specific antibody production due to rank and social instability.

Separate studies examined the influence of the social environment of male cynomolgus macaques on primary and secondary antibody responses to immunization with tetanus toxoid. All animals showed evidence of both primary and secondary anti-tetanus antibody response. In the first study, subordinate animals had a greater primary antibody response to tetanus toxoid, while a single social reorganization (acute stressor) did not influence the response. In the second study, social rank was not associated with the secondary antibody response but repeated social reorganizations (chronic stressor) resulted in a greater level of specific antibody production in comparison to nonreorganized controls. These effects could not be accounted for on the basis of nonspecific differences in total serum IgG or serum albumin.

Soluble interleukin-2 receptor as a marker of lymphocyte activation in childhood Crohn's disease.

Serum concentrations of the soluble form of the interleukin-2 receptor (sIL-2R) were determined by an enzyme-linked immunosorbent assay in a group of 39 pediatric and adolescent patients with Crohn's disease and in age-matched ulcerative colitis patients and controls. sIL-2R levels were found to be elevated in patients with Crohn's disease (p less than 0.001), and increased sIL-2R levels were detected in patients with clinically more severe disease. sIL-2R levels correlated more closely with other laboratory markers of disease activity than with a disease activity score. A progressive increase in sIL-2R levels was noted to correlate with endoscopic measurement of disease extent, while sIL-2R levels did not correlate with other markers of systemic lymphocyte activation, suggesting possible local mucosal production. Sequential determinations in individual patients revealed a good correlation between sIL-2R and clinical course. More important, elevated levels of sIL-2R preceded clinical relapse of asymptomatic patients. We conclude that sIL-2R measurement may be a useful adjunct to clinical assessment and routine laboratory testing in pediatric and adolescent patients with Crohn's disease and that serial levels may be predictive of clinical course and the response to therapy.

Evidence for locally synthesized and clonally restricted immunoglobulin in the synovial fluid from rheumatoid arthritis patients.

In this study, we examined the immunoglobulin (Ig) present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) to determine if it was locally produced and to assess the presence of clonally restricted (oligoclonal) immunoglobulin. We studied SF/serum pairs from 55 RA patients and 23 patients with degenerative joint disease (DJD). We found increases in total protein, IgG, IgA, and IgM in RA vs DJD SF (P less than 0.01). The immunoglobulin present in RA appeared to be locally produced as evidenced by significant increases (P less than 0.01) in the immunoglobulin indices. Regression analysis among the levels of IgG, IgA, and IgM RF and the Ig indices suggested that only a minority of the locally synthesized Ig present was specific for RF. To provide evidence of clonal restriction, we further analyzed the SF specimens by isoelectric focusing and assessed the presence of oligoclonal bands present only in RA SF. In 7/55 RA specimens (13%) we found unique SF IgG bands. All bands were of similar isoelectric point (pI), being quite cathodic with pI greater than 7.5. Our evidence supports synthesis of Ig within RA synovium, with a minority of patients showing prominent and unique SF Ig bands. This suggests an oligoclonal response in SF of some patients, but polyclonal Ig synthesis in most.

Soluble interleukin-2 receptor: elevated levels in serum and synovial fluid of patients with rheumatoid arthritis.

Soluble interleukin-2 receptor (sIL-2R) levels were quantitated in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and degenerative joint disease (DJD). A sandwich immunoassay, employing two monoclonal antibodies against distinct epitopes on the IL-2R, was utilized for measurement. We found a striking elevation of sIL-2R in RA SF as compared with DJD SF (RA, 1319 +/- 135; DJD, 416 +/- 59; p less than 0.001). RA serum sIL-2R levels were also significantly elevated over DJD levels. There was no interaction between rheumatoid factor (RF) and sIL-2R. RA patients with elevated sIL-2R levels had significantly longer disease duration, higher c-reactive protein (CRP) levels in serum and SF, and higher RF levels in serum and SF. The groups were similar in regard to other laboratory variables. The presence of elevated levels of sIL-2R in RA serum and SF confirms the presence of a heightened immune reactivity and in vivo activation of lymphocytes in RA.

Antibasement membrane antibody-mediated experimental conjunctivitis.

In ocular cicatricial pemphigoid, the binding of circulating antibodies to conjunctiva is believed to initiate an antibody-mediated cytotoxic response that results in inflammation and tissue damage. To develop a model of antibody-mediated conjunctival inflammation, we examined the effect on conjunctiva of local or systemic administration of a murine monoclonal antibody against basement membrane of stratified squamous epithelium. Neonatal rabbits were given either a single subconjunctival or intraperitoneal injection of the antibody. Eyes were graded clinically for inflammation and conjunctival biopsies were performed. After subconjunctival injection, clinical and histologic inflammation as well as murine antibody and rabbit complement binding to conjunctival basement membrane were detected. With systemic administration there was post-injection clinical inflammation, and conjunctival basement membrane-bound murine antibody was detected. There was no difference observed in conjunctival mitotic rate or goblet cell frequency between treatment groups and controls, following either route of administration. We have created, therefore, a model for antibody-mediated conjunctivitis in rabbits by local or systemic administration of a monoclonal antibody against a component of stratified squamous epithelial basement membrane.

Elevated soluble CD8 in the synovial fluid from patients with rheumatoid arthritis.

Suppressor/cytotoxic T cells express the surface marker CD8, which can be measured in a soluble form in culture supernatants of activated human lymphocytes. Using a sandwich immunoassay, we assessed the levels of soluble CD8 (sCD8) in serum from patients with rheumatoid arthritis (RA; n = 82), patients with degenerative joint disease (DJD; n = 40), and healthy controls. There were no differences in serum sCD8 levels among these groups. In contrast, the levels of soluble CD8 in the synovial fluid (SF) from patients with RA (n = 53) were significantly increased compared with the levels in 23 samples from patients with DJD (821 +/- 110 U/ml versus 213 +/- 13 U/ml, p less than 0.001). Synovial fluid sCD8 levels in the RA group were strikingly elevated, to a maximum value of 5,026 U/ml. In the majority of RA SF specimens (39 of 53), the values were significantly higher in the SF than the serum. Although the RA group had higher values of sCD8, such values were not significantly correlated with measured laboratory or clinical parameters. Current clinical and laboratory methods of evaluating patients may not be adequate in dealing with the complexity and heterogeneity of RA. Soluble CD8 values may be useful in further grouping patients with this disease.

Measurement of rheumatoid factor isotypes in the clinical laboratory.

In this study we assessed the clinical utility of measuring all major rheumatoid factor (RF) isotypes (IgG, IgA, and IgM) in the diagnostic immunology laboratory using an enzyme-linked immunoassay (ELISA). An improved method for IgG-RF was tested which employed a commercially available monoclonal anti-human IgG Fd antibody and did not require pepsin digestion of samples. We detected elevated levels of all three RF isotypes in a population of hospitalized rheumatoid arthritis patients (n = 109). We demonstrated a significant association between IgM and IgA RF which occurred in 36% of our subjects, while less than 6% had IgM + IgG RF or IgG + IgA RF. A comparison of the IgM ELISA with the Rheumaton revealed a statistically significant correlation (r = 0.65, p = 0.001). In addition, the two methodologies were equivalent in sensitivity (ELISA: 76%, Rheumaton: 78%). However, the ELISA procedure was more time consuming, costly, and required greater technical expertise. The following clinical and laboratory findings were significantly associated with RF isotypes: IgG RF and the presence of rheumatoid nodules (p = 0.03), elevated erythrocyte sedimentation rate (ESR) and IgG RF (p = 0.007), and elevated ESR and IgM RF (p = 0.0009). Our ELISA methodology did not provide significant advantages over existing techniques to justify its use as part of the routine laboratory assessment of rheumatoid factor.

Rheumatoid factors determined by fluorescence immunoassay: comparison with qualitative and quantitative methods.

Fluorescence immunoassay (FIA), a relatively new technique for measuring rheumatoid factors (RF), is automated, quantitative, and calibrated against the Centers for Disease Control reference material for RF. We studied the FIA method in relation to a panel of RF methods, both qualitative [latex (LA) and sheep cell agglutination (SSCA)], and quantitative [nephelometry and enzyme-linked immunoassay (ELISA)]. Regression analysis revealed a highly significant correlation between FIA and either LA (r = 0.90) or nephelometry (r = 0.87). The correlation between FIA and either SSCA (r = 0.62) or ELISA (r = 0.67) was less strong. FIA had the highest sensitivity (91%) of all these methods; the specificity was 86%. FIA provides an accurate, sensitive, and specific measure of RF, and is a good alternative for laboratories wanting to replace titer methods with automated laboratory analysis.

Adult rheumatoid arthritis is associated with MC1, a new HLA-D encoded determinant.

We evaluated the association of a new HLA-D encoded determinant, MC1, with adult rheumatoid arthritis (RA). This determinant associates with DR1 and DR4 and can be defined by serological typing. We found MC1 in 83% of 80 patients with RA vs 43% of controls. Although the frequencies of DR1 and DR4 were both significantly increased in patients with RA compared with controls, MC1 had the highest relative risk (6.2) of any HLA-DR antigen tested. MC1 negative and positive populations were not significantly different in any of a variety of clinical and laboratory variables including age, sex, disease duration, age at onset, hours of morning stiffness, functional class, joint count, presence of subcutaneous nodules or bony erosions, frequency of side effects to gold or D-penicillamine, sedimentation rate, and antinuclear antibody.

The acridine orange fluorochrome microassay: a new technique for quantitation of neutrophil function in burned patients.

A new assay for determination of neutrophil bacterial killing and phagocytosis is presented. The acridine orange (AO) fluorochrome microassay is a simple, reliable technique for assessing polymorphonuclear (PMN) function. It requires small amounts of blood and provides a rapid and reproducible quantitation of neutrophil activity. Using this technique, bacterial killing and phagocytosis were assessed in a group of five severely burned patients admitted to the Medical College of Virginia Burn Unit. All patients studied demonstrated a significant decrease in bacterial killing at some point during their clinical course. The AO assay was found to be a reliable and effective means of quantitating PMN bacterial phagocytosis and killing in this group of burned patients.

Diagnostic procedures in allergy III. Office screening for immune deficiency.

Using the screening tests discussed in this review, the practicing allergist can obtain an adequate assessment of the patient's immune system. Using the clinical nature of the particular patient's infectious history as a guide to the selection of the few simple assays described, the physician can identify those patients who require further evaluation of their immune system. In addition, he can assure those who have concerns about their own or their children's immune system that it is functioning normally.