2.1.

Cells, Viruses, and Treatment

African green monkey kidney (Vero) cells were purchased from the American Type Culture Collection (ATCC), Rockville, Maryland. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 1% glutamine, 50-U/ml penicillin, and 50- $\mathrm{\mu }$ g/ml streptomycin, and incubated at 37°C in humidified air containing 5% ${\mathrm{CO}}_2$ . Vero cells were used for the infection of herpes simplex virus type 1 (HSV-1). HSV-1 was obtained from ATCC (VR-735).

HSV-1 infected cells were treated with various concentrations (0.1, 1, and $10\mathrm{\mu }\mathrm{M}$ ) of the known antiherpetic drug acyclovir (ACV) immediately postinfection (PI). The treatment continued up to the end of the experiment.

Human lymphocytes were isolated from ten healthy persons and ten leukemia patients. Both healthy persons and leukemia patients were 10 to 30 years old. Standard chemotherapy treatment was given to the leukemia patients.¹⁹

2.2.

Cell Infection and Determination of Cytopathic Effect

Monolayers of Vero cells (seeded at $1.5\times {10}^5$ cells/well on 24-well culture plates) were incubated with HSV-1 at the appropriate multiplicity of infection $\left(1\mathrm{MOI}\right)$ in RPMI medium containing 2% FCS at 37°C for $2\mathrm{h}$ . The unabsorbed virus particles were removed, fresh medium containing 2% FCS with or without various concentrations of the antiherpetic drug acyclovir (ACV) was added, and the cell monolayers were incubated at 37°C. At the required time, the CPE in cell culture was examined under an inverted microscope.

2.3.

Isolation of Lymphocytes

Lymphocytes were isolated as previously described.²⁰ Briefly, $3\mathrm{ml}$ of blood was loaded over $3\mathrm{ml}$ of Histopaque (purchased from Sigma, St. Louis, Missouri) solution and centrifuged at $300\mathrm{g}$ for $30\min$ at 23°C. The Histopaque solution is the mixture of Metrizoic acid and Ficoll solution having density of $1.077\mathrm{g}∕\mathrm{ml}$ . The lymphocyte layer (mononuclear cells), located at the middle of the tube, was isolated. The separated lymphocytes were washed twice with $10\mathrm{ml}$ of saline by centrifugation at $300\mathrm{g}$ for $10\min$ at 23°C, and resuspended in $100\mathrm{\mu }$ L of saline solution. The cells were counted with a hematocytometer, and all tested samples were centrifuged again and resuspended in saline solution at a concentration of $1000\mathrm{cells}∕\mathrm{ml}$ .

2.4.

Preparation of Infected and Uninfected Vero Cells for Examination by Fourier Transform Infrared Microscopy

Infected and uninfected Vero cell monolayers were washed twice with saline and the cells were picked up from the tissue culture plates after treatment with trypsin (0.25%) for $1\min$ . The cells were pelleted by centrifugation at $1000\mathrm{rpm}$ for $5\min$ . Each pellet was washed twice with saline and resuspended in $100\mathrm{\mu }$ l of saline. The number of cells was counted with a hematocytometer, and all tested samples were pelleted again and resuspended in an appropriate volume of saline to give a concentration of $1000\mathrm{cells}∕\mathrm{\mu }$ l.

2.5.

Preparation of Slides for Examination by Fourier Transform Infrared Microscopy

Since ordinary glass slides exhibit strong absorption in the wavelength range of interest to us, we used zinc selenide crystals, which are highly transparent to IR radiation. A drop of $1\mathrm{\mu }$ l of each sample (lymphocytes or Vero cell suspensions) was placed on the zinc selenide crystal, air dried for $4\mathrm{h}$ , and examined by FTIR microscopy. The radius of such a 1- $\mathrm{\mu }$ l drop was about $1\mathrm{mm}$ .

2.6.

Fourier Transform Infrared Spectra

FTIR measurements were performed in transmission mode with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector of FTIR microscope (Bruker IRScope II) coupled to the FTIR spectrometer (Bruker Equinox model 55/S, Opus software) (Ettlingen, Germany). The spectra were obtained in the wavenumber range of 600 to $2000{\mathrm{cm}}^{-1}$ in the mid-IR region. A spectrum was taken as an average of 128 scans to increase the signal-to-noise ratio (SNR), and the spectral resolution was $4{\mathrm{cm}}^{-1}$ . The aperture used in this study was $100\mathrm{\mu }\mathrm{m}$ , since we found that this aperture gives the best SNR. At lower apertures, the quality of spectra was bad due to high levels of noise. The cells in the chosen apertures are homogeneous without impurities (such as salts, media components, etc.) and the number of cells in such apertures is about 100. Baseline correction and normalization were performed for all the spectra by Opus software. Baseline correction was done by rubberband method. For the construction of the baseline, the spectrum is divided up in $n$ ranges of equal size. In each range, the minimum $y$ value is determined. The baseline is created by connecting minima with straight lines. Starting from “below,” a rubberband is stretched over this curve. The rubber band is the baseline. The baseline points that do not lie on the rubberband are discarded. Normalization was done by vector method. The average $y$ value of the spectrum is calculated first. This average value is then subtracted from the spectrum so that the middle of the spectrum is pulled down to $y=0$ . The sum of the squares of all $y$ values is then calculated, and the spectrum is divided by the square root of this sum. The vector norm of the result spectrum is 1. Peak positions were determined using the second derivation method by Opus software. For each cell type, the spectrum was taken as the average of five different measurements at various sites of the sample.

Each experiment with each cell type was repeated five times and 25 measurements were obtained from each sample. It is important to mention that there are no considerable differences in spectra from various sites of the same sample; standard deviation (SD) did not exceed 0.005.

2.7.

Cluster Analysis

Among the various mathematical methods applied for classification in biology and medicine, cluster analysis is one of the simplest and most rapid procedures.²¹ In our study, this technique was used to classify certain regions of the FTIR spectra of the examined samples. Cluster analysis was performed by Opus software with a standard making-distance matrix and Ward’s algorithm dendrogram.

3.1.

Fourier Transform Infrared Spectra of Infected Cells with Herpes Simplex Virus Type 1

Vero cells were infected with HSV-1 and examined by FTIR microscopy at 24 and $48\mathrm{h}$ postinfection (PI), and their spectral behavior was examined by FTIR microscopy. The results presented in Fig. 1a showed the FTIR spectra of both control and infected Vero cells at 24 and $48\mathrm{h}$ PI. In general, the infected cells show higher intensities of absorbance at various regions of the scanned area. For both normal and HSV infected cells, the dominant bands at 1655 and $1546{\mathrm{cm}}^{-1}$ were attributed to protein amide 1 and 2 bands.¹ The shoulder at about $1730{\mathrm{cm}}^{-1}$ was attributed to lipid $\mathrm{C}\mathrm{O}$ stretching vibrations.¹ The band at $1465{\mathrm{cm}}^{-1}$ was assigned to the ${\mathrm{CH}}_2$ bending mode of the cell lipids. The bands at 1454 and $1397{\mathrm{cm}}^{-1}$ were attributed to asymmetric and symmetric ${\mathrm{CH}}_3$ bending modes of end ethyl groups and branched methyl groups of proteins and lipids, respectively.²² The peaks at 1237 and $1082{\mathrm{cm}}^{-1}$ were attributed to asymmetric and symmetric stretching vibrations and phospholipids.^{1, 23} The peak at $1064{\mathrm{cm}}^{-1}$ resulted from the overlap of several bands, including absorption due to the vibrational modes of $-{\mathrm{CH}}_2\mathrm{OH}$ and the $\mathrm{C}\text{-}\mathrm{O}$ stretching vibration coupled to the $\mathrm{C}\text{-}\mathrm{O}$ bending mode of cell carbohydrates.²⁴ The peak at $857{\mathrm{cm}}^{-1}$ was attributed to N-type sugars.²⁵

Fig. 1

(a) FTIR spectra in the region 600 to $2000{\mathrm{cm}}^{-1}$ of control Vero cells, with cells infected with HSV-1 at 24- and 48-h PI. (b) FTIR microspectroscopy in the region 1250 to $1350{\mathrm{cm}}^{-1}$ for the tested cells. Data are means of five different and separate experiments for each cell culture.

A considerable gradual absorption increase in the region 1250 to $1350{\mathrm{cm}}^1$ at the various examined times PI was observed in all infected cell cultures [Fig. 1b]. Furthermore, the position of the peak at $857{\mathrm{cm}}^{-1}$ in noninfected control cells was gradually shifted to about $854{\mathrm{cm}}^{-1}$ in infected cells, in correlation with the development of the viral infection (Table 1 ). These parameters were used for follow up after the development of the viral infection in the next experiments of this study.

Table 1

Changes in peak position at 857cm−1 in HSV-1 infected Vero cells treated or untreated with various concentrations of ACV.

3.2.

Treatment of Herpes Simplex Virus Type 1 Infected Vero Cells with Antiherpetic Drug Acyclovir

Vero cells were infected with HSV-1 and treated immediately PI with increasing concentrations of ACV. The FTIR spectra obtained at 48-h PI of these cells show spectral intensity values at regions 1250 to $1350{\mathrm{cm}}^1$ lower than those of the infected nontreated cells but slightly higher than the control uninfected cells, according to the used concentrations of ACV (Fig. 2 ). At concentrations of 1 and $10\mathrm{\mu }$ M of ACV, the spectral results of the infected and treated cells demonstrated only a slight increase compared to the control uninfected cells, while at the lower used concentration of $0.1\mathrm{\mu }$ M, there is a further increase in the spectral intensity values at this region (Fig. 2). The results presented in Table 1 show only a moderate shift of peak at 857- ${\mathrm{cm}}^{-1}$ position in infected and treated cells compared to the infected nontreated cells. This shift is also dependent on the concentration of the used ACV, while it is smaller in the higher drug concentrations.

Fig. 2

(a) FTIR spectra in the region 1250 to $1350{\mathrm{cm}}^{-1}$ of control Vero cells, with cells infected with HSV-1 at 48-h PI, cells infected with HSV-1 and treated with 0.1- $\mathrm{\mu }$ M ACV at 24-h PI, and cells infected with HSV-1 and treated with 0.1- $\mathrm{\mu }$ M ACV at 48-h PI. (b) Same as (a) but treated with 1- $\mathrm{\mu }$ M ACV. (c) Same as (a) but treated with 10- $\mathrm{\mu }$ M ACV. Data are means of five different and separate experiments for each cell culture.

In addition, the results presented in Fig. 2 show the possibility of follow up after the effect of the used drug as a function of time PI. It can be seen from these results that at 24-h PI, the spectra of the infected and treated cells with 1 and $10\mathrm{\mu }$ M of ACV in regions 1250 to $1350{\mathrm{cm}}^{-1}$ are fully similar to the control uninfected cells, while the infected untreated cells show significantly higher values of the spectra. At this point of time, it can be seen that in infected cells treated with $0.1\mathrm{\mu }$ M of ACV, the spectral values already start to increase. At $48\mathrm{h}$ , there is only a slight increase in the spectral levels of cells infected and treated with either 1 or $10\mathrm{\mu }$ M of ACV, compared to the control uninfected cells, while in those treated with $0.1\mathrm{\mu }$ M, there is a notable increase in the spectra levels. Full and excellent classification of all infected and treated samples with $1\mathrm{\mu }$ M of ACV compared to the control infected and untreated samples was obtained in the region 1280 to $1350{\mathrm{cm}}^{-1}$ by cluster analysis (Fig. 3 ).

Fig. 3

Cluster analysis of the IR spectra of the examined control uninfected and HSV-1 infected treated or untreated cells with 1- $\mathrm{\mu }$ M ACV in the frequency range 1280 to $1350{\mathrm{cm}}^{-1}$ . This cluster analysis was made on results obtained at 48-h PI. Five samples from each kind of cell, control uninfected (CONT), infected (HSV-1), and infected and treated with ACV (ACV) were examined.

These results are in agreement with the observed morphological changes [cytopathic effect (CPE) development] of the infected cells (Fig. 4 ), although spectral changes are detectable significantly earlier than morphological changes. The results in Fig. 4 show that the CPE is observed in the infected nontreated cells only 48-h PI, while the spectroscopic changes are measured earlier at $24\mathrm{h}$ , as shown in Fig. 1. Also, in the case of infected and treated cells with the various concentrations of ACV, low levels of CPE were observed only at 72 to 90-h PI in cells treated with 1 or $10\mathrm{\mu }$ M of ACV and at 48 to $60\mathrm{h}$ in those treated with $0.1\mathrm{\mu }$ M.

Fig. 4

Effect of ACV on the kinetics of CPE development after infection with HSV-1. Vero cells were infected with $1\mathrm{MOI}$ of HSV-1 and treated with various concentrations of ACV immediately PI. CPE was evaluated by inverted microscope observations. Data are means $\pm SD$ $(n=5)$ .

3.3.

Spectral Behavior of Leukemia Lymphocytes Before and After Chemotherapy Treatment

Lymphocytes were isolated from healthy persons and from leukemia patients before and after a course of chemotherapy treatment, and their spectral behavior was examined by FTIR microscopy. Our results show significant and remarkable differences between normal and leukemia lymphocytes (Fig. 5 ). Some of these differences are represented by a significant reduction in the intensity of the absorbance due to a ${\mathrm{PO}}_2\text{-}$ asymmetric stretching band (in the region 1250 to $1350{\mathrm{cm}}^{-1}$ ) for leukemia lymphocytes compared to normal lymphocytes [Fig. 5b].

Fig. 5

(a) FTIR spectra in the region 600 to $2000{\mathrm{cm}}^{-1}$ of lymphocytes obtained from healthy persons, leukemia patients, and leukemia patients after 1 week of treatment with the appropriate drug. (b) FTIR spectra in the region 1250 to $1350{\mathrm{cm}}^{-1}$ of the same cells in (a). (c) FTIR spectra in the region 1050 to $1100{\mathrm{cm}}^{-1}$ of the same cells in (a). Data are means of ten different samples from either healthy persons or leukemia patients.

Also, our measurements show a considerable and detectable shift of the peak at 1082 to $1083{\mathrm{cm}}^{-1}$ (which represent the ${\mathrm{PO}}_2\text{-}$ symmetric stretching band) for the normal lymphocytes 1086 to $1087{\mathrm{cm}}^{-1}$ in leukemia lymphocytes [Fig. 5c].

Our results show that lymphocytes from leukemia patients became very similar to normal lymphocytes in their IR spectra at the end of treatment, as can be seen in Fig. 5. The results presented in Figs. 5b and 5c show that both the position of the ${\mathrm{PO}}_2\text{-}$ symmetric stretching band and the intensity of ${\mathrm{PO}}_2\text{-}$ asymmetric stretching in the treated leukemia lymphocytes goes in the direction of their values in normal lymphocytes.