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AIM To understand the interference of carbohydrates absorbance in nucleic acids signals during diagnosis of malignancy using Fourier transform infrared (FTIR) spectroscopy. METHODS We used formalin fixed paraffin embedded colonic tissues to obtain infrared (IR) spectra in the mid IR region using a bruker II IR microscope with a facility for varying the measurement area by varying the aperture available. Following this procedure we could measure different regions of the crypt circles containing different biochemicals. Crypts from 18 patients were measured. Circular crypts with a maximum diameter of 120 μm and a lumen of about 30 μm were selected for uniformity. The spectral data was analyzed using conventional and advanced computational methods. RESULTS Among the various components that are observed to contribute to the diagnostic capabilities of FTIR, the carbohydrates and nucleic acids are prominent. However there are intrinsic difficulties in the diagnostic capabilities due to the overlap of major absorbance bands of nucleic acids, carbohydrates and phospholipids in the mid-IR region. The result demonstrates colonic tissues as a biological system suitable for studying interference of carbohydrates and nucleic acids under ex vivo conditions. Among the diagnostic parameters that are affected by the absorbance from nucleic acids is the RNA/DNA ratio, dependent on absorbance at 1121 cm-1 and 1020 cm-1 that is used to classify the normal and cancerous tissues especially during FTIR based diagnosis of colonic malignancies. The signals of the nucleic acids and the ratio (RNA/DNA) are likely increased due to disappearance of interfering components like carbohydrates and phosphates along with an increase in amount of RNA. CONCLUSION The present work, proposes one mechanism for the observed changes in the nucleic acid absorbance in mid-IR during disease progression (carcinogenesis).

Spectroscopic techniques have been finding increasing applications in the field of biomedicine especially in the field of disease diagnosis and monitoring in spite of the rapid emergence of several...

Lupus nephritis is a devastating complication of systemic lupus erythematosus (SLE) for which current therapies are insufficiently effective. Histologic evaluation of renal biopsies is a poor predictor of therapeutic response or outcome. Integrated immunologic, genomic and proteomic approaches may yield new insights into disease pathogenesis and thereby improve therapeutic strategies for lupus nephritis. Given the lack of sequential biopsies from humans, it also remains essential to study informative animal models of disease. Cross-species analyses can identify cells or pathways that are relevant to human disease and can be further studied in mouse models. Using a systems biology approach in which we compare molecular data from kidneys of three different mouse models of lupus nephritis with data from human lupus biopsies, we have found that inflammatory events escalate rapidly around the time of proteinuria onset. This is followed by hypoxia and metabolic stress, and by tubular and endothelial dysfunction. The failure of complete reversal of these abnormalities may increase the sensitivity of the kidney to further insult. We further found that renal macrophages and dendritic cells are key players in lupus nephritis both in mouse models and humans and that macrophages display a hybrid molecular profile that reflects incomplete resolution of inflammation and excessive tissue remodeling. Finally, our studies have suggested several new biomarkers for disease stage that can now be tested longitudinally in human SLE patients.

Kidney involvement affects 40–60% of patients with lupus, and is responsible for significant morbidity and mortality. Using depletion approaches, several studies have suggested that macrophages may play a key role in the pathogenesis of lupus nephritis. However, “off target” effects of macrophage depletion, such as altered hematopoiesis or enhanced autoantibody production, impeded the determination of a conclusive relationship. In this study, we investigated the role of macrophages in mice receiving rabbit anti-glomerular antibodies, or nephrotoxic serum (NTS), an experimental model which closely mimics the immune complex mediated disease seen in murine and human lupus nephritis. GW2580, a selective inhibitor of the colony stimulating factor-1 (CSF-1) receptor kinase, was used for macrophage depletion. We found that GW2580-treated, NTS challenged mice did not develop the increased levels of proteinuria, serum creatinine, and BUN seen in control-treated, NTS challenged mice. NTS challenged mice exhibited significantly increased kidney expression of inflammatory cytokines including RANTES, IP-10, VCAM-1 and iNOS, whereas GW2580-treated mice were protected from the robust expression of these inflammatory cytokines that are associated with lupus nephritis. Quantification of macrophage related gene expression, flow cytometry analysis of kidney single cell suspensions, and immunofluorescence staining confirmed the depletion of macrophages in GW2580-treated mice, specifically within renal glomeruli. Our results strongly implicate a specific and necessary role for macrophages in the development of immune glomerulonephritis mediated by pathogenic antibodies, and support the development of macrophage targeting approaches for the treatment of lupus nephritis.

Objective To characterize renal macrophages and dendritic cells (DCs) in 2 murine models of lupus nephritis. Methods We used a bead-based enrichment step followed by cell sorting to isolate populations of interest from young mice and nephritic mice. Cell morphology was examined by microscopy. Arginase and nitrite production was examined using biochemical assays. The antigen-presenting functions of the cells were determined using mixed lymphocyte reactions. Selected cytokine, chemokine, and Toll-like receptor (TLR) profiles were examined using real-time quantitative polymerase chain reaction. Results We identified 2 populations of macrophages and 3 populations of DCs in both of our murine models of lupus (NZB/NZW and [NZW × BXSB]F1 mice). F4/80high macrophages, which were resident in normal kidneys and found to be increased in number during nephritis, did not produce either arginase or nitrite upon cytokine stimulation and acquired a mixed proinflammatory and antiinflammatory functional phenotype during nephritis that resembles the constitutively activated phenotype of gut F4/80high macrophages. The various cell types differed in their expression of chemokine receptors and TLRs, consistent with variability in their renal location. Resident renal CD103+ DCs were the best antigen-presenting cells and could easily be distinguished from CD11chigh myeloid DCs that accumulated in large numbers during nephritis. Conclusion Our study highlights the heterogeneity of the macrophage/DC infiltrate in chronic lupus nephritis and provides an initial phenotypic and functional analysis of the different cellular components that can now be used to define the role of each cell subset in nephritis progression or amelioration. Of note, the dominant macrophage population that accumulates during nephritis has an acquired phenotype that is neither M1 nor M2 and may reflect failure of resolution of inflammation.

SummaryA hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.

Renal infiltration with mononuclear cells is associated with poor prognosis in systemic lupus erythematosus. A renal macrophage/dendritic cell signature is associated with the onset of nephritis in NZB/W mice, and immune-modulating therapies can reverse this signature and the associated renal damage despite ongoing immune complex deposition. In nephritic NZB/W mice, renal F4/80hi/CD11cint macrophages are located throughout the interstitium, whereas F4/80lo/CD11chi dendritic cells accumulate in perivascular lymphoid aggregates. We show here that F4/80hi/CD11cint renal macrophages have a Gr1lo/Ly6Clo/VLA4lo/MHCIIhi/CD43lo/CD62Llo phenotype different from that described for inflammatory macrophages. At nephritis onset, F4/80hi/CD11cint cells upregulate cell surface CD11b, acquire cathepsin and matrix metalloproteinase activity, and accumulate large numbers of autophagocytic vacuoles; these changes reverse after the induction of remission. Latex bead labeling of peripheral blood Gr1lo monocytes indicates that these are the source of F4/80hi/CD11cint macrophages. CD11chi/MHCIIlo dendritic cells are found in the kidneys only after proteinuria onset, turnover rapidly, and disappear rapidly after remission induction. Gene expression profiling of the F4/80hi/CD11cint population displays increased expression of proinflammatory, regulatory, and tissue repair/degradation-associated genes at nephritis onset that reverses with remission induction. Our findings suggest that mononuclear phagocytes with an aberrant activation profile contribute to tissue damage in lupus nephritis by mediating both local inflammation and excessive tissue remodeling.

The process of carcinogenesis in the colon progresses through several overlapping stages, making the evaluation process challenging, as well as subjective. Owing to the complexity of colonic tissues and the search for a technique that is rapid and foolproof for precise grading and evaluation of biopsies, many spectroscopic techniques have been evaluated in the past few decades for their efficiency and clinical compatibility. Fourier-transform infrared spectroscopy, being quantitative and objective, has the capacity for automation and relevance to cancer diagnosis. This article highlights investigations on the application of Fourier-transform infrared spectroscopy (particularly microscopy) in colon cancer diagnosis and parallel developments in data analysis techniques for the characterization of spectral signatures of malignant tissues in the colon.

Fourier transform infrared (FTIR)-spectroscopy has been found useful for monitoring the effectiveness of drugs during chemotherapy in leukemia patients. In the present work, spectral changes that occurred in the white blood cells (WBC) of an adult acute myeloid leukemia (AML) patient and their possible utilization for monitoring biochemistry of WBC were investigated. The phosphate absorbance from nucleic acids and the lipid-protein ratio in the WBC decreased immediately after treatment and then increased to levels of a control group. Similar observations were recorded in child patients with acute lymphoblastic leukemia (ALL) who were used as test cases. These parameters maybe used as possible markers to indicate successful remission and suggest that FTIR-spectroscopy may provide a rapid optical method for continuous monitoring or evaluation of a WBC population.

Fourier transform infrared (FTIR) spectroscopy has shown remarkable ability in distinguishing between bacterial species and identifying bacterial colony structures, when used in tandem with methods such as cluster analysis, principal component analysis, or linear discriminant analysis. The present work was aimed to evaluate the potential of FTIR–microscopy (FTIR-MSP) to distinguish between different serotypes and capsular quantities of Streptococcus pneumoniae. In general, the results obtained have consistently proven that the spectral information at the region 900–1185 cm−1 was sufficient to distinguish between various pneumococcal serotypes. Moreover, the method was able to differentiate between S. pneumoniae phase variants on the basis of their relative carbohydrate content. The unsupervised cluster analysis of the samples showed differences, not only in the carbohydrate content, but also in the region 1350–1480 cm−1, which is dominated by absorptions due to lipids and phospholipids. This approach proved to be useful for the distinction between S. pneumoniae serotypes and between phase variants, which were shown to acquire different pathogenic capacity. © 2006 Wiley Periodicals, Inc. Biopolymers 83:434–442, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Detection of malignancy at early stages is crucial in cancer prevention and management. Fourier transform infrared (FTIR) spectroscopy has shown promise as a non-invasive method with diagnostic potential in cancer detection. Studies were conducted with formalin-fixed biopsies of melanoma and cervical cancer by FTIR microspectroscopy (FTIR-MSP) to detect common biomarkers, which occurred in both types of cancer distinguishing them from the respective non-malignant tissues. Both types of cancer are diagnosed on skin surfaces. The spectra were analysed for changes in levels of biomolecules such as RNA, DNA, phosphates and carbohydrate (glycogen). Whereas carbohydrate levels showed a good diagnostic potential for detection of cervical cancer, this was not the case for melanoma. However, variation of the RNA/DNA ratio as measured at I(1121)/I(1020) showed similar trends between non-malignant and malignant tissues in both types of cancer. The ratio was higher for malignant tissues in both types of cancer.

Recently, Fourier transform infrared (FTIR)-spectroscopy has been used to monitor cell growth by several works. Conventionally, the study of cell and tissue dynamics at molecular levels is carried out through various approaches like histochemical methods, application of molecular biology and immunology. Colonic crypts display a pattern in cell growth along their height. Histologically normal sections obtained from formalin fixed biopsies of colon cancer patients were studied in the present work through vibrational spectroscopy. The evolution and development of the normal human colonic crypts manifested in Fourier transform infrared-microspectroscopy (FTIR-MSP) as spectral changes in the levels of nucleic acids, proteins, carbohydrates and lipids. The results indicate that the level of carbohydrates, nucleic acids and lipids increases only till the middle of the crypt up to which the maturation zone is restricted and thereafter decreases till the top where the cells are exfoliated. These observations are in coherence with earlier reports on crypt proliferation. We identify the normal pattern of various biochemicals along the colonic crypt based on data analyzed from FTIR-MSP. This study affords an important example of the application of microscopic vibrational spectroscopy for understanding basic cell processes from formalin fixed tissues where in vivo studies and immunological methods are not feasible.

Chemokines facilitate the recruitment of inflammatory cells into tissues, contributing to target organ injury in a wide range of inflammatory and autoimmune diseases. Targeting either single chemokines or chemokine receptors alters the progression of disease in animal models of rheumatoid arthritis and lupus with varying degrees of efficacy, but clinical trials in humans have been less successful. Given the redundancy of chemokine-chemokine receptor interactions, targeting of more than one chemokine may be required to inhibit active inflammatory disease. To test the effects of multiple chemokine blockade in inflammation, we generated an adenovirus expressing bovine herpesvirus 1 glycoprotein G (BHV1gG), a viral chemokine antagonist that binds to a wide spectrum of murine and human chemokines, fused to the fragment crystallizable (Fc) portion of murine immunoglobulin (IgG)2a. Administration of the adenovirus significantly inhibited thioglycollate-induced migration of polymorphonuclear leukocytes into the peritoneal cavity of BALB/c mice and reduced both clinical severity and articular damage in K/BxN serum transfer-induced arthritis. However, treatment with BHV1gG-Ig fusion protein did not prevent monocyte infiltration into the peritoneum in the thioglycollate model and did not prevent renal monocyte infiltration or nephritis in lupus-prone NZB/W mice. These observations suggest that the simultaneous inhibition of multiple chemokines by BHV1gG has the potential to interfere with acute inflammatory responses mediated by polymorphonuclear leukocytes, but is less effective in chronic inflammatory disease mediated by macrophages.

Components present in the acellular fraction of blood influence the blood cell survival and function and the response to biotic and abiotic factors. Human plasma and sera have been used as therapeutic agents and are known to increase cell survival. White blood cells in normal blood are exposed to plasma components in vivo, but the effect of such plasma components in vitro on adherent peripheral blood mononuclear cells (PBMCs) that includes monocytes has not been fully investigated. We cultured human PBMCs with autologous plasma and observed structural variation due to plasma addition in PBMCs along with increased cell survival. Light microscopy of the cells showed increased granularity in plasma-treated cells. Fourier transform infrared (FTIR) spectroscopy was used to elucidate the possible mechanism by studying the changes in the biochemical composition of the cells that explained the observations. FTIR spectroscopy of plasma-treated cells show altered spectral pattern in the mid-IR region, indicating increased phospholipid levels. Heat-stable components in the plasma possibly increase the differentiation of PBMCs, as evident by increased phospholipid metabolism. The data suggest that plasma-stimulated membrane biogenesis may contribute to PBMC survival by inducing them to differentiate into antigen presenting cells (APCs) like macrophages and dendritic cells.

To characterize renal macrophages and dendritic cells (DCs) in 2 murine models of lupus nephritis.We used a bead-based enrichment step followed by cell sorting to isolate populations of interest from young mice and nephritic mice. Cell morphology was examined by microscopy. Arginase and nitrite production was examined using biochemical assays. The antigen-presenting functions of the cells were determined using mixed lymphocyte reactions. Selected cytokine, chemokine, and Toll-like receptor (TLR) profiles were examined using real-time quantitative polymerase chain reaction.We identified 2 populations of macrophages and 3 populations of DCs in both of our murine models of lupus (NZB/NZW and [NZW × BXSB]F1 mice). F4/80(high) macrophages, which were resident in normal kidneys and found to be increased in number during nephritis, did not produce either arginase or nitrite upon cytokine stimulation and acquired a mixed proinflammatory and antiinflammatory functional phenotype during nephritis that resembles the constitutively activated phenotype of gut F4/80(high) macrophages. The various cell types differed in their expression of chemokine receptors and TLRs, consistent with variability in their renal location. Resident renal CD103+ DCs were the best antigen-presenting cells and could easily be distinguished from CD11c(high) myeloid DCs that accumulated in large numbers during nephritis.Our study highlights the heterogeneity of the macrophage/DC infiltrate in chronic lupus nephritis and provides an initial phenotypic and functional analysis of the different cellular components that can now be used to define the role of each cell subset in nephritis progression or amelioration. Of note, the dominant macrophage population that accumulates during nephritis has an acquired phenotype that is neither M1 nor M2 and may reflect failure of resolution of inflammation.

Cancer diagnosis using infrared spectroscopy uses absorbance of nucleic acids, carbohydrates and phosphates as biomarkers. The present work deciphers the mutual intereference in absorbance of these compnents using colonic tissues as a model.

Fourier transform infrared spectroscopy was used to evaluate the effects of autologus human plasma on peripheral blood mononuclear cells with a view to understanding the biochemical changes associated with increased survival

Male NZW/BXSB.Yaa (W/B) mice express two copies of TLR7 and develop pathogenic autoantibodies, whereas females with only one copy of TLR7 have attenuated disease. Our goal was to analyze the regulation of the autoantibody response in male and female W/B mice bearing the autoreactive site-directed H chain transgene 3H9. Serum anti-dsDNA Abs appeared in males at 12 wk, and most had high-titer IgG anti-dsDNA and anti-cardiolipin Abs and developed >300 mg/dl proteinuria by 8 mo. Females had only low-titer IgG anti-cardiolipin Abs, and none developed proteinuria by 1 y. Males had a smaller marginal zone than females with a repertoire that was distinct from the follicular repertoire, indicating that the loss of marginal zone B cells was not due to diversion to the follicular compartment. Vk5-43 and Vk5-48, which were rare in the naive repertoire, were markedly overrepresented in the germinal center repertoire of both males and females, but the VJ junctions differed between males and females with higher-affinity autoreactive B cells being selected into the germinal centers of males. Administration of IFN-α to females induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a male pattern of junctional diversity in Vk5-43 and Vk5-48. Our studies are consistent with the hypothesis that presence of the Yaa locus, which includes an extra copy of Tlr7, or administration of exogenous IFN-α relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the Ag-driven B cell repertoire.

In this chapter we present methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans. Using magnetic beads followed by flow cytometric sorting, pure populations of cells are obtained that are functional in a variety of assays. Sufficient numbers of cells are obtained for genomic characterization. An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that cause renal impairment in SLE and other renal inflammatory diseases.

TNFα (TNF) is an important regulator of physiologic and inflammatory immune responses. Although TNF antagonists have remarkable therapeutic benefits in several autoimmune diseases including rheumatoid arthritis, psoriasis and Crohn’s disease, they also have been associated with the development of anti-nuclear antibodies and even clinical SLE, multiple sclerosis and other demyelinating diseases. TNF is protective in the early stages of SLE in some mouse models but is overexpressed in inflamed target organs in both mice and humans with SLE (1–2). Consistent with both protective and pro-inflammatory roles of TNF, blockade of TNF improved proteinuria in patients with refractory SLE nephritis but increased anti-DNA and anti-cardiolipin autoantibody titers (3). In this study, we determined the clinical effects of TNFR2-Ig in a mouse model of lupus nephritis in which an increase in serum levels of TNF and renal production of TNF occurs concomitantly with the onset of renal disease. TNF inhibition introduced after the development of autoantibodies and at the onset of clinical nephritis stabilized the nephritis and resulted in a long period of relapsing and remitting proteinuria that was associated with a marked improvement in survival. Mechanistic studies revealed that there was no effect of TNF inhibition on autoantibody production or lymphocyte activation in the spleen or renal autoantibody deposition, however there was a marked decrease in renal periglomerular and interstitial accumulation of F4/80hi renal macrophages. Real-time PCR analysis revealed a significant decrease in renal expression of chemokines CCL2, CCL5 and CCL9, the endothelial activation marker VCAM-1, genes involved in tissue remodeling and the markers of proximal tubule damage lipocalin-2 and HAVCR1 (KIM-1). We conclude that TNF inhibition decreases the renal inflammatory response to immune complex deposition. This is associated with decreased accumulation of periglomerular and interstitial renal macrophages that play a role in tissue remodeling, and decreased damage of renal tubular cells. In contrast, perivascular aggregates containing CD11chi dendritic cells as well as B and T cells accumulate in the kidneys in a TNF independent manner and are not sufficient to induce terminal renal damage.

Biochemical analyses of substances rely upon the ability of techniques to identify qualitatively and quantitatively the components present and are based on physicochemical characteristics as well as chemical nature of substances being detected. While chemical analyses usually depend on reactions of a given substance and can be destructive, spectral studies are usually non-destructive and deal with describing a substance based on properties like absorption or transmission of light (e.g. UV, Visible, Infrared (IR)), light scattering ability, fluorescence /phosphorescence using various optical techniques. Thus, the technique (Fourier transform infrared) spectroscopy has gained prominence in both research and applications in different fields of science. Among the various techniques, IR spectroscopy owing to its lower potency of causing damage compared to X-rays, gamma rays and UV rays (as it is based on weak vibrational energies) has become the technique of choice during chemical analysis of substances. IR spectroscopy can not only provide information about the various components in a complex material but is also unique in its ability to be modified into different kinds of instrumentations based on requirement. The various IR spectroscopy based instruments from a simple IR based spectroscope that helps to obtain the absorbance spectra of a chemical compound to the complex imaging systems that employ computational methods in addition to the technical sophistication are based on a simple principle that every compound or a particular combination of compounds can be described by means of a FTIR (Fourier transform infra red) spectra qualitatively and quantitatively.

In this chapter we present methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans. Using magnetic beads followed by flow cytometric sorting, pure populations of cells are obtained that are functional in a variety of assays. Sufficient numbers of cells are obtained for genomic characterization. An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that cause renal impairment in SLE and other renal inflammatory diseases.

Background Recent advances in chemotherapeutic treatment of childhood acute leukemia have improved remission rates to about 80%. With the development of novel drugs and treatment protocols adapted for specific individual patients, a simple diagnostic tool for following patients' responses on a daily basis is required. In the present clinical study, we have investigated the usefulness of Fourier transform infrared microscopy (FTIR-MSP) for pre-screening and follow-up of leukemia patients undergoing chemotherapy. Methods Blood samples were collected from leukemia patients before and during treatment as well as from patients with high fever and healthy subjects which served as control groups. Peripheral blood mononuclear cells (PBMCs) were isolated and their spectra obtained using FTIR-MSP. The presence of blasts in bone marrow and other diagnostic and prognostic clinical parameters were determined during follow-up up to 1000 days. Results Leukemia was efficiently indicated by a reduced lipids and elevated DNA absorption of PBMC together with additional characteristic spectral bands. These diagnostic markers were used for monitoring the biochemical changes in PBMCs during chemotherapy. The trends of several markers were found to be in agreement with blast percentage as determined by flow cytometry. Conclusions Our findings reveal the utility of FTIR-MSP for leukemia pre-screening independently of symptoms common to leukemia. Furthermore, FTIR-MSP supplies precursor indication regarding patient response to treatment compared to current methods. General significance This preliminary study shows a great potential of FTIR-MSP as a complementary tool for childhood leukemia pre-screening and follow-up which may allow faster response to critical problems arise during treatment.

Immunosenescence is characterized by a decreased ability of the immune system to respond to foreign antigens, as well as a decreased ability to maintain tolerance to self-antigens. This results in an increased susceptibility to infection and cancer and reduced responses to vaccination [1-5]. The mechanisms underlying immunosenescence comprise a series of cellular and molecular events involving alteration of several biochemical pathways and different cellular populations, and for the most part our understanding of these molecular mechanisms is still fragmentary. In this review we will focus on the process of senescence associated with oxidative stress, in particular how protein oxidation alters the functionality of immune cells and how oxidative stress contributes to a chronic inflammatory process often referred as inflamm-aging.

This study aimed to determine the potential of IR-spectroscopy to diagnose abnormality in histologically normal resection margins for predicting relapse in colon cancer patients. The present study evaluates potential abnormal crypt proliferation in histologically normal resection margins. Resection margins of 10 colon cancer patients (adenocarcinoma) (27 biopsies in all), found completely normal by standard histology were re-evaluated. Fourier transform infrared microscopy (FTIR-MSP) was performed on the longitudinal sections of the crypt, and spectral data collected from the base, middle and top portion of crypts. Absorbance in the region 900-1185 cm(-1) arising from carbohydrates and nucleic acids was found to be the most effective variate for such evaluation. In total 225 crypts were classified after assessing the levels of abnormality observed by the above technique. The abnormal biopsies detected using the above optical method was correlated with a relapse in the patient's history. Patients who had a relapse had at least one abnormal biopsy (crypt) based on the present methodology. Among the patients, the only case without a relapse was also the case where no abnormal crypts were found in any biopsies from the resection margins. The agreement between the biopsy status, as determined by the optical methodology, and the relapse of colonic malignancy based on the patients' medical files, establishes the translational nature of FTIR-MSP for medical purposes and hints at future clinical evaluation of the biopsies using this technique to determine more precisely the zone of excision during anastomosis.

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H-ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H-ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H-ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 x 10(6) cells/ml whereas for transformed cells it was up to 1.4 x 10(6) cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering.

Fourier transform infrared microspectroscopy (FTIR-MSP) is potentially a powerful analytical method for identifying the spectral properties of biological activity in cells. The goal of the present research is the implementation of FTIR-MSP to study early spectral changes accompanying malignant transformation of cells. As a model system, cells in culture are infected by the murine sarcoma virus (MuSV), which induces malignant transformation. The spectral measurements are taken at various postinfection time intervals. To follow up systematically the progress of the spectral changes at early stages of cell transformation, it is essential first to determine and validate consistent and significant spectral parameters (biomarkers), which can evidently discriminate between normal and cancerous cells. Early stages of cell transformation are classified by an array of spectral biomarkers utilizing cluster analysis and discriminant classification function techniques. The classifications indicate that the first spectral changes are detectable much earlier than the first morphological signs of cell transformation. Our results point out that the first spectral signs of malignant transformation are observed on the first and third day of postinfection (PI) (for NIH/3T3 and MEF cell cultures, respectively), while the first visible morphological alterations are observed only on the third and seventh day, respectively. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent-free method for early detection of malignancy.

Colorectal cancer is one of the most aggressive cancers usually occurring in people above the age of 50 years. In the United States, colorectal cancer is the third most diagnosed cancer. The American Cancer Society has estimated 96,830 new cases of colon cancer and 40,000 new cases of rectal cancer in 2014 in the United States. According to the literature, up to 55% of colorectal cancer patients experience a recurrence within five years from the time of surgery. Relapse of colorectal cancer has a deep influence on the quality of patient life. Infrared (IR) spectroscopy has been widely used in medicine. It is a noninvasive, nondestructive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer. Abnormalities in the colonic crypts, which are not detectable using standard histopathological methods, could be determined using IR spectroscopic methods. The IR measurements were performed on formalin-fixed, paraffin-embedded colorectal tissues from eight patients (one control, four local recurrences, three distant recurrences). A total of 128 crypts were measured. Our results showed the possibility of differentiating among control, local, and distant recurrence crypts with more than a 92% success rate using spectra measured from the crypts' middle sites.

TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa) cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa) or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

The rapid developments in the field of infrared spectroscopy in the past decade have demonstrated a potential for disease diagnosis using noninvasive technologies. Several earlier studies have highlighted the advantage of using infrared spectroscopy both in the near- and mid-infrared regions for diagnostic purposes at clinical levels. The areas of focus have been the distinction of premalignant and malignant cells and tissues from their normal state using specific parameters obtained from Fourier transform infrared spectra, making it a rapid and reagent-free method. While it still requires pilot studies and designed clinical trials to ensure the applicability of such systems for cancer diagnosis, substantial progress has been made in incorporating advances in computational methods into the system to increase the sensitivity of the entire setup, making it an objective and sensitive technique suitable for automation to suit the demands of the medical community. The development of fiber-optics systems for infrared spectroscopy have further opened up new and modern avenues in medical diagnosis at various levels of cells, tissues and organs under laboratory and clinical conditions.

Fourier transform infrared microspectroscopy (FTIR-MSP) has shown promise as a technique for detection of abnormal cell proliferation and premalignant conditions. In the present study, we investigate the absorbance in the sensitive wavenumber region between 2800 and 3000 cm(-1), which has been known to be due to the antisymmetric and symmetric stretching vibrations of CH2 and CH3 groups of proteins and lipids. We report common biomarkers from this region that distinguish between normal and malignant tissues and cell lines. Based on our findings, we propose that the wavenumber region around 2800 to 3000 cm(-1) in the FTIR spectra of cells and tissues could provide valuable scientific evidence at the onset of premalignancy and may be used for ex vivo and in vitro detection of carcinogenesis. To further examine the utility of these markers in cancer diagnosis and management, they are tested successfully in monitoring the changes occurring in leukemia patients during chemotherapy.

Malignant melanoma, a malignant neoplasm of epidermal melanocytes is the third most common skin cancer. In many cases, melanoma develops from nevus, which is considered as the nonmalignant stage. Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between melanoma, nevus, and the corresponding normal epidermis. In the present work, FTIR-MSP was performed on formalin-fixed biopsies of melanoma and nevi along with the adjoining histologically normal epidermis to understand the biochemical variations from the epidermis and identify suitable parameters for differentiation of nevi from melanoma. The comparative analysis of various parameters calculated from the spectral data of the normal epidermis and the abnormal regions showed that the changes in the nucleic acids was a significant indicator of the abnormal nature of the tissues. The RNA/DNA ratio was decreased in case of both melanoma and nevus compared to the epidermis. The amide II/amide I ratio was greater for nevus and melanoma compared to the epidermis. In contrast to other organs, the analysis of carbohydrates was not found as a suitable indicator in case of malignant melanoma. Shifts in band wave number were found to be a major distinguishing feature between the melanoma and compound nevi. The present study helps in the identification of spectral features suitable for distinction of melanoma from nevus that appear similar even in FTIR spectral features and thus can pave the way for development ofin vivoscreening systems based on these diagnostic markers.

FTIR spectroscopy has been extensively used to understand the differences between normal and malignant cells and tissues. In the present study, FTIR microspectroscopy was performed on biopsies to evaluate parameters deduced from changes in nucleic acid absorbance monitored at various characteristic wavenumbers in the Mid-IR region. The data showed that there were differences in the spectra of normal and malignant tissues from several organs such as colon, cervix, skin and blood with respect to absorbance due to nucleic acids. Similar results were observed in the case of cell lines that were transformed to induce carcinogenesis. Of the several ratios examined for consistency in differentiating cancer and normal tissues, the I(996 cm−1)/I(966 cm−1) showed promise as a distinguishing parameter and was comparable to the I(1121 cm−1)/I(1020 cm−1) ratio reported in many earlier studies. The absorbance of nucleic acids is presented with an emphasis on the application of FTIR microspectroscopy for diagnosis of malignancy. Our results indicate that usage of nucleic acid absorbance yield statistically significant parameters, which could differentiate normal and cancerous tissues.

Abnormal crypt proliferation and development in the colon has been associated with premalignant stages of colon cancer. Conventionally, molecular markers are used to detect abnormal crypt proliferation.In the present work, feasibility studies of FTIR-MSP to distinguish between normal and abnormal crypts from colon biopsies that show normal histopathological features have been undertaken.The results indicate that abnormal crypts show deviations in the pattern of absorbance in the Mid IR region along the crypt height when compared with the normal crypts. The crypts could be empirically classified into three groups such as crypts having a normal absorbance pattern for all biochemical components, crypts with abnormal absorbance pattern for some biochemical components and crypts with completely abnormal absorbance pattern along the height for all or most biochemical components studied by FTIR. The utilization of FTIR-MSP is proposed for diagnosis of abnormal metabolism at the molecular level of histologically completely normal-looking crypts, especially from those biopsies that are taken from sites far away from cancer.This method could give rise to a reduction in false-negative results during examination of biopsies using the conventional histopathological methods. The present method may be complementary to existing methods for precise demarcation of the zone of colostomy prior to colon cancer surgery.

The early diagnosis and proper identification of cervical squamous intraepithelial lesions plays an important role in a good prognosis for the patient. However, the present practice of screening based on PAP (Papanicolaou) smear and histopathology makes it tedious and prone to human errors. We assess the validity of FTIR microspectroscopy (FTIR-MSP) of biopsies as a method to properly assign the correct stage of premalignancy in patients with symptoms of cervical intraepithelial neoplasia. For the first time we evaluate the biopsies based on the FTIR spectra for different grades of neoplasia in tandem with probabilistic neural networks (PNNs) and histopathology. The results show that the grading of neoplasia based on FTIR-MSP and a PNN differentiates the normal from premalignant with a high level of accuracy. The false positive identification of the normal as cervical intraepithelial neoplasia 1 (CIN1), CIN2, and CIN3 patients is 9.04, 0.01, and 0.01%, respectively. The false negative identification of CIN2 patients as normal and CIN1 patients is 0.01 and 4.4%, respectively. Similarly, the false negative identification of CIN3 patients as normal, CIN1, and CIN2 is 0.14, 6.99, and 9.61%, respectively. The small errors encountered in the grading are comparable to current methods, encouraging advanced studies for the development of mechanized equipment for the diagnosis and grading of premalignant cervical neoplasia.

A novel methodology for the diagnosis of acute infections using FTIR microspectroscopy (FTIR-MSP) data on blood components and cluster analysis is presented. Blood samples were collected from 11 patients suffering from various infections and 16 age-matched healthy human controls. Blood components such as white blood cells, red blood cells, and plasma were isolated using standard procedures and FTIR-MSP of these components was utilized. A cluster analysis of the FTIR spectra was performed. The spectra obtained from the three blood components of patients were different from those of controls. The FTIR spectra of white blood cells from patients suffering infections were significantly different from the controls. Cluster analyses of averaged FTIR-MSP spectra of white blood cells provided 100% classification between patients and healthy controls.

Mice were inoculated intranasally with Streptococcus pneumoniae isolates of serotype 14 with different genetic backgrounds (14R, 14DW) and a capsular switch of 14R, strain 9VR (serotype 9V). Inoculation of the mice with 14R and 9VR resulted in 60% mortality. All the mice survived 14DW inoculation. No differences in lungs' bacterial loads were found 3 h following inoculation. Bacterial clearance of 5 logs was observed 48 h after inoculation with 14DW versus within 1 log 48 h after inoculation with 14R and 9VR. No significant differences in bacterial size or the capsular amount could be found between 14R and 14DW. We conclude that factor(s) in addition to the capsule, contribute to disease outcome.

Autophagy is process by which cells recycle cellular components to overcome starvation. This process is constitutive in antigen presenting cells such as dendritic cells and macrophages. Among the various autophagy pathways, microautophagy (Mi) has a unique significance as it is implicated in the biogenesis of exosomes which are relevant to the design of therapeutics. Statins have been shown to up regulate macroautophagy and alleviate the effect of LPS apart from inhibiting cholesterol synthesis. However their role in microautophagy has not been reported. In the present study we investigated the effect of statins on microautophagy using the RAW264.7 cell line as a model system. Treatment with statins resulted in an up regulation of the genes for MA as well as Mi as determined by qPCR. Further studies showed that the levels of proteins TSG101 and VPS4 were increased during statin treatment. Pretreatment with statins led to a lowering of LAMP2A levels and decreased LAMP2A on the late endosomes, mimicking a LAMP2A knock down. Pretreatment with statins not only lowered the transcription of iNOS and TNF-alpha genes but also prevented the effect of LPS by decreasing initial levels of LAMP2A and increasing levels of TSG101. We propose that Mi can be a surrogate for Chaperone mediated autophagy (CMA) under conditions where the components of CMA are rendered non-functional due to disruption in normal cellular metabolism.

Background The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing. Methodology/Principal Findings A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. Conclusions/Significance The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance.

Infrared spectroscopy is widely perceived as a future technology for cancer detection and grading. Malignant melanoma, an aggressive skin cancer, is accessible to non-invasive IR radiation based surface probes for its identification and grading. The present work examines the differences in the IR spectra of melanoma tissues and the surrounding epidermis in skin biopsies with the objective of identifying diagnostic parameters and suitable computational/statistical methods of analysis. Melanoma could be differentiated from the epidermis in biopsies of 55 patients, using parameters derived from absorbance bands originating from molecular vibrations of nucleic acids and/or their bases. Additionally, absorbances from tyrosine and phosphate that are abnormally elevated in malignant melanoma could be used as markers. Two-dimensional plots of these parameters in tandem with advanced statistical methods successfully demonstrate the potential of IR spectroscopy to distinguish between epidermal and melanoma regions with a high classification success. The work underlines the importance of developing data analysis methods in FTIR based diagnosis using melanoma as a model system.

Fourier-transform infrared spectroscopy has shown alterations of spectral characteristics of cells and tissues as a result of carcinogenesis. The research reported here focuses on the diagnosis of cancer in formalin-fixed biopsied tissue for which immunochemistry is not possible and when PAP-smear results are to be confirmed. The data from two groups of patients (a control group and a group of patients diagnosed with cervical cancer) were analyzed. It was found that the glucose/phosphate ratio decreases (by 23-49%) and the RNA/DNA ratio increases (by 38-150%) in carcinogenic compared with normal tissue. Fourier-transform microspectroscopy was used to examine these tissues. This type of study in larger populations may help to set standards or classes with which to use treated biopsied tissue to predict the possibility of cancer. Probabilistic neural networks and statistical tests as parts of these biopsies predict the possibility of cancer with a high degree of accuracy (> 95%).

SummaryAutophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVBs). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways.