Fourier transform infrared spectroscopy as a non-destructive method for analysing herbarium specimens.

Dried plant specimens stored in herbaria are an untapped treasure chest of information on environmental conditions, plant evolution and change over many hundreds of years. Owing to their delicate nature and irreplaceability, there is limited access for analysis to these sensitive samples, particularly where chemical data are obtained using destructive techniques. Fourier transform infrared (FTIR) spectroscopy is a chemical analysis technique which can be applied non-destructively to understand chemical bonding information and, therefore, functional groups within the sample. This provides the potential for understanding geographical, spatial and species-specific variation in plant biochemistry. Here, we demonstrate the use of mid-FTIR microspectroscopy for the chemical analysis of Drosera rotundifolia herbarium specimens, which were collected 100 years apart from different locations. Principal component and hierarchical clustering analysis enabled differentiation between three main regions on the plant (lamina, tentacle stalk and tentacle head), and between the different specimens. Lipids and protein spectral regions were particularly sensitive differentiators of plant tissues. Differences between the different sets of specimens were smaller. This study demonstrates that relevant information can be extracted from herbarium specimens using FTIR, with little impact on the specimens. FTIR, therefore, has the potential to be a powerful tool to unlock historic information within herbaria.

Profile of BAFF and its receptors' expression in lupus nephritis is associated with pathological classes.

Background/Objective B-cell activating factor (BAFF) plays an important role in the pathogenesis of systemic lupus erythematosus. However, the role of BAFF in lupus nephritis (LN) is not understood. Our aim was to evaluate the expression of BAFF and its three receptors in renal biopsy samples from patients with LN and investigate a relationship with pathological class. Methods We conducted a prospective descriptive study (2011-2014) on 52 kidney biopsy samples from patients with LN. Immunohistochemistry for BAFF, its receptors (transmembrane activator and calcium modulator and cyclophilin ligand interaction (TACI), protein maturation of B cells (BCMA), and BAFF-receptor (BAFF-R)), and CD20 expression was performed. Samples were scored according to the percentage of cells with positive expression. Results In class II LN, BAFF-R and TACI were not expressed, whereas BCMA and BAFF were lowly expressed in the interstitial inflammatory infiltrates. Proliferative class III/IV had elevated BAFF expression in the glomeruli, and TACI was expressed in interstitial inflammatory infiltrates and the glomeruli. Interestingly, the class IV cases with vasculopathy ( n = 4) had endothelial BAFF expression, which was not visible in thrombotic microangiopathy ( n = 4). Class V was characterized by low BAFF expression in interstitial inflammatory infiltrates and by BAFF, TACI, and BCMA expression in the glomeruli. BAFF expression was associated with inflammatory scores and CD20 positive infiltrates, mainly in class IV. Conclusions Expression patterns of BAFF and its receptors differ according to LN class. Our study provides evidence that BAFF could be used as a routine marker in LN biopsies and to determine which patients will benefit from anti-BAFF therapy.

Vibrational spectroscopic monitoring and biochemical analysis of pericellular matrix formation and maturation in a 3-dimensional chondrocyte culture model.

Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce a cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 µm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain the chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimicking hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogel's capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses of the key components of the PCM. For the first time, synchrotron based Fourier transform infrared (SR-FTIR) microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. In contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAGs than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions of the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to the chondron samples, while the day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocyte and chondron samples, but showed that the cultured chondrocyte samples at day 7, day 14 and day 21 clustered together. These data would indicate that SR-FTIR microspectroscopy can help to better understand the PCM formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Fourier transform infrared spectra of cells on glass coverslips. A further step in spectral pathology.

Over the last few years, great effort has been placed on developing Fourier Transform Infrared (FTIR) microspectroscopy as a tool to help in the histopathological diagnosis of cancer. The ever increasing workload in pathology departments is calling for a technique that could identify the presence of cancer cells in cytology and tissue samples in an objective, fast and automated way. However, pathologists use glass slides which absorb infrared (IR) radiation thus removing important mid-IR spectral data in the fingerprint region (proteins, DNA, RNA; 1800 cm-1 to 900 cm-1). To this purpose, we hypothesised whether using thinner glass slides, i.e., glass coverslips, would allow us to obtain spectral data not only from the lipid region (3100 cm-1 to 2700 cm-1) but also from the fingerprint region. To this purpose, we studied peripheral blood mononuclear cells (PBMC), a leukaemia cell line (K562) and a lung cancer cell line (CALU-1). Cells were placed on DAKO coverslips and their FTIR spectra obtained at MIRAS beamline, Alba synchrotron light source (Barcelona, Catalonia). The data presented here not only shows for the first time that it is possible to obtain spectral data from most of the amide I region (1800 cm-1 to 1570 cm-1) of cells placed on glass coverslips but more important, principal component analysis was able to separate between the three types of cells for both the lipid and the amide I regions. The methodology here described is a further step in the application of FTIR microspectroscopy in histopathology departments.

Effects of nilotinib on leukaemia cells using vibrational microspectroscopy and cell cloning.

Over the last few years, both synchrotron-based FTIR (S-FTIR) and Raman microspectroscopies have helped to better understand the effects of drugs on cancer cells. However, cancer is a mixture of cells with different sensitivity/resistance to drugs. Furthermore, the effects of drugs on cells produce both chemical and morphological changes, the latter could affect the spectra of cells incubated with drugs. Here, we successfully cloned sensitive and resistant leukaemia cells to nilotinib, a drug used in the management of leukaemia. This allowed both the study of a more uniform population and the study of sensitive and resistant cells prior to the addition of the drug with both S-FTIR and Raman microspectroscopies. The incubation with nilotinib produced changes in the S-FTIR and Raman spectra of both sensitive and resistant clones to nilotinib. Principal component analysis was able to distinguish between cells incubated in the absence or presence of the drug, even in the case of resistant clones. The latter would confirm that the spectral differences between the so-called resistant clonal cells prior to and after adding a drug might reside on those more or less sensitive cells that have been able to remain alive when they were collected to be studied with S-FTIR or Raman microspectroscopies. The data presented here indicate that the methodology of cell cloning can be applied to different types of malignant cells. This should facilitate the identification of spectral biomarkers of sensitivity/resistance to drugs. The next step would be a better assessment of sensitivity/resistance of leukaemia cells from patients which could guide clinicians to better tailor treatments to each individual patient.

Worsening of calcinosis cutis with teriparatide treatment in two osteoporotic patients.

We present two cases of patients with systemic autoimmune diseases (one with dermatomyositis and one with CREST syndrome) who presented with a worsening of calcinosis cutis after treatment of osteoporosis with teriparatide. To our knowledge, this association is not described in the literature and might be considered in the spectrum of adverse reactions to teriparatide.

Dielectric spectroscopy of normal and malignant human lung cells at ultra-high frequencies.

Microwave techniques for biomedical applications aimed at cancer treatment or diagnosis, either by imaging or spectroscopy, are promising. Their use relies on knowledge of the dielectric properties of tissues, especially on a detectable difference between malignant and normal tissues. As most studies investigated the dielectric properties of ex vivo tissues, there is a need for better biophysical understanding of human tissues in their living state. As an essential component of tissues, cells represent valuable objects of analysis. The approach developed in this study is an investigation at cell level. Its aim was to compare human lung normal and malignant cells by dielectric spectroscopy in the beginning of the microwave range, where such information is of substantial biomedical importance. These cells were embedded in small and low-conductivity agarose hydrogels and laid on an open-ended coaxial probe connected to a vector network analyser operated from 200 MHz to 2 GHz. The comparison between normal and malignant cells was drawn using the variation of measured dielectric properties and fitting the measurements using the Maxwell-Wagner equation. Both methods revealed slight differences between the two cell lines, which were statistically significant regarding conductivities of composite gels and cells.

Molecular subtyping reveals immune alterations associated with progression of bronchial premalignant lesions.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

Estimating and correcting mie scattering in synchrotron-based microscopic fourier transform infrared spectra by extended multiplicative signal correction.

We present an approach for estimating and correcting Mie scattering occurring in infrared spectra of single cells, at diffraction limited probe size, as in synchrotron based microscopy. The Mie scattering is modeled by extended multiplicative signal correction (EMSC) and subtracted from the vibrational absorption. Because the Mie scattering depends non-linearly on alpha, the product of the radius and the refractive index of the medium/sphere causing it, a new method was developed for estimating the Mie scattering by EMSC for unknown radius and refractive index of the Mie scatterer. The theoretically expected Mie contributions for a range of different alpha values were computed according to the formulae developed by Van de Hulst (1957). The many simulated spectra were then summarized by a six-dimensional subspace model by principal component analysis (PCA). This subspace model was used in EMSC to estimate and correct for Mie scattering, as well as other additive and multiplicative interference effects. The approach was applied to a set of Fourier transform infrared (FT-IR) absorbance spectra measured for individual lung cancer cells in order to remove unwanted interferences and to estimate ranges of important alpha values for each spectrum. The results indicate that several cell components may contribute to the Mie scattering.

[Pneumococcal conjugate vaccine failure in HIV-infected child. Clinical case]. / Fallo de la vacuna antineumocócica conjugada en un niño VIH positivo. A propósito de un caso.

Despite appropriate antimicrobial therapy and vaccination, invasive pneumococcal infections remain associated with significant mortality, especially in selected high-risk groups (asplenic, humoral immunity deficient patients, etc.). We present a 13-year-old caucasian boy with HIV infection (vertical transmission). He received treatment with highly-active antiretroviral therapy (amprenavir, lamivudine and zidovudine) and vaccination with 23-valent vaccine (6 years old) and 7-valent pneumococcal conjugate vaccine (10 years old). His CD4 count and his viral load at these times were 2,063/microl and 13461 cop/ml, when he was 6 years old and 1,315/microl and 32400 cop/ml when he was 10 years old, respectively. The latest CD4 count (1,000/microl) and his viral load (3800 cop/ml) confirmed satisfactory control of the disease. He was referred to our emergency department presenting with fever, head and stomach-ache and vomiting. In the following hours his condition continued to deteriorate and depressed level of consciousness and meningismus were observed. Streptococcus pneumoniae, serotype 18 C, was detected in blood and cerebrospinal fluid cultures. Despite appropriate treatment with antibiotics (cefotaxime and vancomycin) and anti-oedema medications, brain-death was confirmed 24 hours after his admittance.

Cellular bioenergetics after erythropoietin therapy in chronic renal failure.

After erythropoietin (rHuEPO) therapy, patients with chronic renal failure (CRF) do not improve peak O2 uptake (VO2 peak) as much as expected from the rise in hemoglobin concentration ([Hb]). In a companion study, we explain this phenomenon by the concurrent effects of fall in muscle blood flow after rHuEPO and abnormal capillary O2 conductance observed in CRF patients. The latter is likely associated with a poor muscle microcirculatory network and capillary-myofiber dissociation due to uremic myopathy. Herein, cellular bioenergetics and its relationships with muscle O2 transport, before and after rHuEPO therapy, were examined in eight CRF patients (27 +/- 7.3 [SD] yr) studied pre- and post-rHuEPO ([Hb] = 7.8 +/- 0.7 vs. 11.7 +/- 0.7 g x dl-1) during an incremental cycling exercise protocol. Eight healthy sedentary subjects (26 +/- 3.1 yr) served as controls. We hypothesize that uremic myopathy provokes a cytosolic dysfunction but mitochondrial oxidative capacity is not abnormal. 31P-nuclear magnetic resonance spectra (31P-MRS) from the vastus medialis were obtained throughout the exercise protocol consisting of periods of 2 min exercise (at 1.67 Hz) at increasing work-loads interspersed by resting periods of 2.5 min. On a different day, after an identical exercise protocol, arterial and femoral venous blood gas data were obtained together with simultaneous measurements of femoral venous blood flow (Qleg) to calculate O2 delivery (QO2leg) and O2 uptake (VO2leg). Baseline resting [phosphocreatine] to [inorganic phosphate] ratio ([PCr]/[Pi]) did not change after rHuEPO (8.9 +/- 1.2 vs. 8.8 +/- 1.2, respectively), but it was significantly lower than in controls (10.9 +/- 1.5) (P = 0.01 each). At a given submaximal or peak VO2leg, no effects of rHuEPO were seen on cellular bioenergetics ([PCr]/[Pi] ratio, %[PCr] consumption halftime of [PCr] recovery after exercise), nor in intracellular pH (pHi). The post-rHuEPO bioenergetic status and pHi, at a given VO2leg, were below those observed in the control group. However, at a given pHi, no differences in 31P-MRS data were detected between post-rHuEPO and controls. After rHuEPO, at peak VO2, Qleg fell 20% (P < 0.04), limiting the change in QO2leg to 17%, a value that did not reach statistical significance. The corresponding O2 extraction ratio decreased from 73 +/- 4% to 68 +/- 8.2% (P < 0.03). These changes indicate that maximal O2 flow from microcirculation to mitochondria did not increase despite the 50% increase in [Hb] and explain how peak VO2leg and cellular bioenergetics (31P-MRS) did not change after rHuEPO. Differences in pHi, possibly due to lactate differences, between post-rHeEPO and controls appear to be a key factor in the abnormal muscle cell bioenergetics during exercise observed in CRF patients.

Maximal lipidic power in high competitive level triathletes and cyclists.

OBJECTIVE: To describe the fat-oxidation rate in triathlon and different modalities of endurance cycling. METHODS: 34 endurance athletes (15 male triathletes, 4 female triathletes, 11 road cyclists and 4 male mountain bikers) underwent a progressive cycloergometer test until exhaustion. Relative work intensity (VO(2max)), minimal lactate concentration (La(-)(min)), lactic threshold, individual lactic threshold (ILT), maximal fat-oxidation rate (Fat(max), Fat(max) zone) and minimal fat-oxidation rate (Fat(min)) were determined in each of the groups and were compared by means of one-way analysis of variance. RESULTS: No significant differences were found for Fat(max), Fat(min) or for the Fat(max) zone expressed as fat oxidation rate (g/min). Intensities -20%, -10% and -5% Fat(max) were significantly lower for mountain bikers with respect to road cyclists and female triathletes, expressed as % VO(2max). Intensities 20%, 10% and 5% Fat(max) were significantly lower for mountain bikers with respect to male triathletes and female triathletes, and for male triathletes in comparison with female triathletes, expressed as % VO(2max). Lactic threshold and La(-)(min) did not show significant differences with respect to Fat(max). Lactic threshold was found at the same VO(2max) with respect to the higher part of the Fat(max) zone, and La(-)(min) at the same VO(2max) with respect to the lower part of the Fat(max) zone. CONCLUSIONS: The VO(2max) of Fat(max) and the Fat(max) zone may explain the different endurance adaptations of the athletes according to their sporting discipline. Lactic threshold and La(-)(min) were found at different relative work intensities with respect to those of Fat(max) even though they belonged to the Fat(max) zone.

Detecting the Presence and Progression of Premalignant Lung Lesions via Airway Gene Expression.

Purpose: Lung cancer is the leading cause of cancer-related death in the United States. The molecular events preceding the onset of disease are poorly understood, and no effective tools exist to identify smokers with premalignant lesions (PMLs) that will progress to invasive cancer. Prior work identified molecular alterations in the smoke-exposed airway field of injury associated with lung cancer. Here, we focus on an earlier stage in the disease process leveraging the airway field of injury to study PMLs and its utility in lung cancer chemoprevention.Experimental Design: Bronchial epithelial cells from normal appearing bronchial mucosa were profiled by mRNA-Seq from subjects with (n = 50) and without (n = 25) PMLs. Using surrogate variable and gene set enrichment analysis, we identified genes, pathways, and lung cancer-related gene sets differentially expressed between subjects with and without PMLs. A computational pipeline was developed to build and test a chemoprevention-relevant biomarker.Results: We identified 280 genes in the airway field associated with the presence of PMLs. Among the upregulated genes, oxidative phosphorylation was strongly enriched, and IHC and bioenergetics studies confirmed pathway findings in PMLs. The relationship between PMLs and squamous cell carcinomas (SCC) was also confirmed using published lung cancer datasets. The biomarker performed well predicting the presence of PMLs (AUC = 0.92, n = 17), and changes in the biomarker score associated with progression/stability versus regression of PMLs (AUC = 0.75, n = 51).Conclusions: Transcriptomic alterations in the airway field of smokers with PMLs reflect metabolic and early lung SCC alterations and may be leveraged to stratify smokers at high risk for PML progression and monitor outcome in chemoprevention trials. Clin Cancer Res; 23(17); 5091-100. ©2017 AACR.

Oncogenic Characterization and Pharmacologic Sensitivity of Activating Fibroblast Growth Factor Receptor (FGFR) Genetic Alterations to the Selective FGFR Inhibitor Erdafitinib.

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3-TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717-26. ©2017 AACR.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Phase I Dose-Escalation Study of JNJ-42756493, an Oral Pan-Fibroblast Growth Factor Receptor Inhibitor, in Patients With Advanced Solid Tumors.

PURPOSE: JNJ-42756493 is an orally administered pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor. This first-in-human study evaluates the safety, pharmacokinetics, and pharmacodynamics and defines the recommended phase II dose (RP2D) of JNJ-42756493. PATIENTS AND METHODS: Eligible patients with advanced solid tumors received escalating doses of JNJ-42756493 from 0.5 to 12 mg administered continuously daily or JNJ-42756493 10 or 12 mg administered intermittently (7 days on/7 days off). RESULTS: Sixty-five patients were enrolled. The most common treatment-emergent adverse events included hyperphosphatemia (65%), asthenia (55%), dry mouth (45%), nail toxicity (35%), constipation (34%), decreased appetite (32%), and dysgeusia (31%). Twenty-seven patients (42%) experienced grade ≥ 3 treatment-emergent adverse events, and one dose-limiting toxicity of grade 3 ALT elevation was observed at 12 mg daily. Maximum-tolerated dose was not defined. Nine milligrams daily was considered as the initial RP2D; however, tolerability was improved with intermittent schedules, and 10 mg administered on a 7-days-on/7-days-off schedule was considered the final RP2D. Pharmacokinetics were linear, dose proportional, and predictable, with a half-life of 50 to 60 hours. Dose-dependent elevations in serum phosphate, a manifestation of pharmacodynamic effect, occurred in all patients starting at 4 mg daily. Among 23 response-evaluable patients with tumor FGFR pathway alterations, four confirmed responses and one unconfirmed partial response were observed in patients with glioblastoma and urothelial and endometrial cancer (all with FGFR2 or FGFR3 translocations); 16 patients had stable disease. CONCLUSION: JNJ-42756493 administered at 10 mg on a 7-days-on/7-days-off schedule achieved exposures at which clinical responses were observed, demonstrated pharmacodynamic biomarker activity, and had a manageable safety profile.

A human melanoma cell line transduced with an interleukin-4 gene by a retroviral vector releases biologically active IL-4 and maintains the original tumor antigenic phenotype.

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10(5) cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.

Interleukin-2 gene-transduced human melanoma cells efficiently stimulate MHC-unrestricted and MHC-restricted autologous lymphocytes.

Two human melanoma lines were transduced by a retroviral vector with the gene of the human interleukin-2 (IL-2) and characterized for their immunological properties in comparison with the parental lines. Transduction resulted in the production of biologically active IL-2 in the average amounts of 2,282 and 2,336 pg/ml per 10(5) cells per 24 hr over 3 and 2 months by the Me14932/IL-2 and the Me1B6/IL-2 lines, respectively. Melanoma-transduced cells lost their tumorigenicity in nude mice. No major changes in the phenotype were observed in IL-2 gene-transduced lines. In fact, more than 90% of cells expressed class I and II(DR) HLA, adhesion molecules, integrins, and melanoma-associated antigens. Irradiation with 100-400 Gy, while inhibiting tumor cell growth in vitro, allowed the release of IL-2 by the transduced cells for at least 5 weeks. The two melanoma lines also maintained susceptibility to lysis by lymphokine-activated killer (LAK) cells and by a HLA-A2-restricted melanoma-specific cytotoxic T lymphocyte (CTL) clone recognizing the melanoma antigen (Melan-A). In a limiting dilution assay, transduced, but not parental melanoma lines unless added with an amount of IL-2 comparable to that released by the transduced cells, were able to expand both nonspecific and melanoma-specific CTL precursors from autologous peripheral blood lymphocytes (PBL). In mixed lymphocytes-tumor cultures, IL-2 gene-transduced melanoma cells stimulated the expansion of major histocompatibility complex (MHC)-unrestricted effectors from autologous PBL, and of CD3+ CD8+ MHC-restricted CTL from tumor-invaded lymph nodes. These results indicate that IL-2 gene transduction does not alter significantly the expression of the immunologically relevant molecules of human melanoma lines while increasing their ability to stimulate both specific and nonspecific lymphocyte responses. These lines will be of value in the vaccination of melanoma patients.

Limited antitumor T cell response in melanoma patients vaccinated with interleukin-2 gene-transduced allogeneic melanoma cells.

We have immunized advanced melanoma patients with a HLA-A2-compatible human melanoma line genetically modified to release interleukin-2 (IL-2), to elicit or increase a T cell-mediated anti-melanoma response that may affect distant lesions. Twelve stage-IV patients were injected subcutaneously at days 1, 13, 26, and 55 with IL-2 gene-transduced and irradiated melanoma cells at doses of 5 or 15 x 10(7) cells. Both local and systemic toxicities were mild, consisting of transient erythema at the vaccination site; fever occurred in a minority of patients. Three mixed responses were recorded. Seven patients were evaluable for immunological studies. Mixed tumor-lymphocyte cultures carried out with different allogeneic HLA-A2-matched melanoma lines as stimulators and targets revealed an increase in the MHC-unrestricted, but no changes in the MHC-restricted, cytotoxicity in peripheral blood lymphocytes (PBL) obtained after vaccination as compared with those obtained before vaccination. Increased recognition of the tyrosinase 368-376 peptide occurred in post-vaccination PBL of one patient, whereas a weak increase in recognition of the gp100 280-288 peptide was detectable in another patient; these 2 patients also recognized the gp100 457-466 peptide. After in vitro, stimulation with the only available autologous melanoma line, CD4+ cells with autologous tumor-specific cytotoxicity and ability to release interferon-gamma (IFN-gamma) were found in post- but not in pre-vaccination PBL. In the same patient, as well as in another patient, limiting dilution analysis showed that vaccination resulted in an increased frequency of melanoma-specific cytotoxic T lymphocyte (CTL) precursors. These results indicate that vaccination with cells releasing IL-2 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although this response occurred in a minority of the melanoma patients studied.

Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells.

A human melanoma line genetically modified to release interleukin 4 (IL-4) was utilized to immunize advanced melanoma patients in order to elicit or increase a specific anti-melanoma immune response, which may affect distant lesions. Twelve metastatic melanoma patients were injected subcutaneously at least three times with 5 x 10(7) IL-4 gene-transduced and irradiated allogeneic melanoma cells per dose. Both systemic and local toxicities were mild, consisting of transient fever and erythema, swelling, and induration at the vaccination site. Two mixed but not complete or partial clinical responses were recorded. To assess the immune response of vaccinated patients, both serological and cell-mediated activities were evaluated. Antibodies to alloantigens could be detected in 2 of 11 patients tested. Mixed tumor-lymphocyte cultures were performed, utilizing autologous and allogeneic HLA-A2-matched melanoma lines as simulators and targets. A significant increase in IFN-gamma release was detected in 7 of 11 cases when postvaccination lymphocytes were stimulated by the untransduced allomelanoma cells. However, induction of a specific recognition of autologous melanoma cells by PBLs was obtained after vaccination in only one of six cases studied. This response involved the melanoma peptide Melan-A/MART-1(27-35) that was recognized in an HLA-A2-restricted fashion. These results indicate that vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although in a minority of patients.