Marc G. Golightly

Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi.

The diagnosis of Lyme disease often depends on the measurement of serum antibodies to Borrelia burgdorferi, the spirochete that causes this disorder. Although prompt treatment with antibiotics may abrogate the antibody response to the infection, symptoms persist in some patients. We studied 17 patients who had presented with acute Lyme disease and received prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently developed. Although these patients had clinically active disease, none had diagnostic levels of antibodies to B. burgdorferi on either a standard enzyme-linked immunosorbent assay or immunofluorescence assay. On Western blot analysis, the level of immunoglobulin reactivity against B. burgdorferi in serum from these patients was no greater than that in serum from normal controls. The patients had a vigorous T-cell proliferative response to whole B. burgdorferi, with a mean ( +/- SEM) stimulation index of 17.8 +/- 3.3, similar to that (15.8 +/- 3.2) in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of both groups was greater than that of a control group of healthy subjects (3.1 +/- 0.5; P less than 0.001). We conclude that the presence of chronic Lyme disease cannot be excluded by the absence of antibodies against B. burgdorferi and that a specific T-cell blastogenic response to B. burgdorferi is evidence of infection in seronegative patients with clinical indications of chronic Lyme disease.

Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM‐coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% ± 9% (n = 18) recovery rate and 0.5–35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18–256 CTCs/ml and average of 126 ± 25 (mean ± SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I–III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node‐status and survival of patients with early stage breast cancer. CAM‐captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM‐captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM‐initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.

Lyme borreliosis‐associated encephalopathy

Borrelia burgdorferi infection (Lyme disease) is frequently accompanied by CNS dysfunction. Particularly common is a mild confusional state, the mechanism of which is unknown. Since CNS infection with B burgdorferi is usually accompanied by intrathecal synthesis of specific antibody, we studied CSF in 73 patients referred for presumed CNS Lyme, manifested primarily as this confusional state. Of 30 seropositive patients evaluated, only 5 had intrathecal antibody production. Seven seronegative patients had positive cell-mediated immune responses to B burgdorferi in the peripheral blood none had antibody production in the CSF. Of the remaining 36 patients referred with this diagnosis despite negative serologic studies, none had compelling evidence of CNS infection by this criterion. We conclude that CNS infection with B burgdorferi does occur in a small proportion of seropositive patients with this confusional state but is extremely uncommon among seronegative individuals with this clinical presentation.

In vivo effect of recombinant human granulocyte colony-stimulating factor on phagocytic function and oxidative burst activity in septic neutropenic neonates.

Neutrophil dysfunction may contribute to an increased risk of sepsis in very-low-birth-weight (VLBW) neonates. The current study was designed to determine whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects absolute neutrophil count (ANC), phagocytic function, and oxidative burst in neutropenic VLBW neonates. Fourteen ventilated VLBW neonates were treated with rhG-CSF (10 µg/kg/day × 3 days i.v.). Phagocytic activity and oxidative burst were assessed before and after treatment with rhG-CSF using flow cytometry and fluorescence labeled opsonized Staphylococcus aureus. Control (nonseptic, nonneutropenic, n = 4), preeclamptic neutropenic (PET; nonseptic, n = 5), and septic neutropenic (n = 5) neonates with a gestational age ranging from 24 to 30 weeks were studied. In both PET and septic neonates, posttreatment phagocytosis more than doubled, but did not achieve matching control levels, whereas rhG-CSF treatment maintained the level of the phagocytic activity in the control group. The oxidative burst increased in all groups, but, again, PET and septic groups did not achieve matching control values. These effects occurred independent of a 2- to 12-fold increase in ANC. These results suggest that other disease-specific factors delay full functional recovery even after rhG-CSF treatment. We speculate that PET and septic neonates may remain susceptible to infection due to deficient neutrophil-killing capacity, even though their ANC returns to normal ranges. Augmenting immune function beyond the immediate period of ANC recovery suggests that prophylaxis with rhG-CSF may be an important risk reduction strategy for susceptible VLBW neonates.

Prognostic analysis of invasive circulating tumor cells (iCTCs) in epithelial ovarian cancer

GOALS Circulating tumor cells (CTCs) have been introduced as a biomarker in detecting advanced epithelial ovarian cancer (EOC). The goals are to examine the prevalence of the invasive subpopulation of CTCs (iCTCs) in patients at high risk of EOC and to compare this biomarker to serum CA125. METHODS We used a unique cell adhesion matrix (CAM)-based, functional cell enrichment and identification platform to isolate iCTCs from 129 preoperative patients. We confirmed the identity of iCTCs using positive epithelial (Epi+) markers and negative hematopoietic lineage (HL-) markers. Sensitivity and specificity of the assays were examined and iCTCs/CA125 were correlated with overall survival (OS), progression-free survival (PFS) and clinical parameters. RESULTS We found a 41.2% sensitivity, 95.1% specificity and 77.8% positive predictive value (PPV) of the iCTC assay in detecting patients with stage I and II EOC malignancy, and a 83% sensitivity and 97.3% PPV in detecting all stages of EOC malignancy. However, a positive CA125 test provided weak evidence to detect stage I and II malignancy (61.6% PPV) and all EOC (92.1% PPV), because of its 76.2% specificity. A significantly stronger concordance in OS and PFS of clinical factors (tumor stage, debulking and platinum sensitivity) was noted for elevated iCTCs than for serum CA125. CONCLUSION The CAM-initiated CTC enrichment/identification method enabled the detection of early stage EOC. iCTCs were better correlated with worse OS and PFS, more specific and better PPV than CA125 in detecting EOC malignancy in patients at high risk of EOC.

Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Treatment monitoring of patients with epithelial ovarian cancer using invasive circulating tumor cells (iCTCs)

GOALS Contemporary management of epithelial ovarian cancer (EOC) uses biomarkers to monitor response to therapy. This study evaluates the role of invasive circulating tumor cells (iCTCs) in monitoring EOC treatment in comparison with serum cancer antigen 125 (CA125). METHODS Molecular and microscopic analyses were used to identify seprase and CD44 as tumor progenitor (TP) markers. The iCTC flow cytometry assay was optimized using blood donated by 64 healthy donors, 49 patients with benign abdominal diseases and 123 EOC patients. Serial changes in iCTCs and CA125 were measured in 129 blood and 169 serum samples, respectively, from 31 EOC patients to assess their concordance during therapy and their relationship with risk of progressive disease (PD). RESULTS The assay had 97% specificity and 83% sensitivity for detecting iCTCs in blood of EOC patients. iCTCs were detected in each monitoring patient (31/31, 100%) and in 110 of the 129 blood samples (85.3%). The concordance between changes in iCTCs/CA125 levels and changes in the intervals associated with no evidence of disease (NED) were markedly stronger (specificity: CA125 93.8%; iCTCs 90.6%), whereas increases in iCTCs (79.5%) were more sensitive than increases in CA125 (67.6%) to predict PD or relapse. Among the six patients who had greater than 6 measurements, iCTCs but not CA125 antedated changes in clinical status from PD to NED during and after chemotherapy and predated relapse. CONCLUSION Serial measurements of iCTCs could predict therapeutic responsiveness in 31 EOC patients who underwent standard taxol/carboplatin therapy.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

High dose cyclophosphamide preferentially targets naïve T (CD45/CD4/RA+) cells in CIDP and MS patients.

INTRODUCTION T cells occupy a central role in MS and CIDP pathogenesis. High dose cyclophosphamides in-vivo cytotoxic-effect on circulating memory and naïve T cells is unknown. METHOD Three MS and five CIDP patients received cyclophosphamide (200 mg/kg) for refractory disease. Before and after chemotherapy administration, peripheral blood T-cell subsets were determined. Patients underwent serial neurologic evaluations quarterly. RESULTS Cyclophosphamide uniformly decreased clinical disease activity. Compared to memory T cells, naïve T cells were preferentially eradicated. DISCUSSION Cyclophosphamide effectiveness in autoimmune illness may result from Naïve T-cell destruction, as this compartment may be the source of autoreactive lymphocytes.

Disparity Between Serological Reactivity to Borrelia burgdorferi and Evidence of Past Disease in a High-Risk Group

A prevalence study of past Lyme borreliosis in persons with outdoor occupations was done. Consenting individuals (n = 302) were administered a questionnaire eliciting demographic and occupational data and a clinical history, and were asked to donate a serum specimen for detection of antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and borrelia inhibition assays, and for detection of potentially cross-reactive antibodies. Of 302 individuals, 77 (25%) had reactive antibodies detected by ELISA. Of these 302 individuals, 44 (15%) met the criteria of the Centers for Disease Control and Prevention for serological reactivity as evidenced by immunoblotting, and 70 (23%) had inhibitory activity. Through the clinical criteria employed, only 11 individuals with serological reactivity had prior illness compatible with Lyme borreliosis. Higher ELISA absorbances were positively correlated with age and duration of outdoor occupation. The results from three serological assays and the lack of reactivity to potentially cross-reactive infectious agents indicate that serological reactivity was due to exposure to B. burgdorferi. The disparity between serological reactivity and the clinical evidence of Lyme borreliosis suggests cumulative exposure to a nonpathogenic form of B. burgdorferi.