VOL. 42 NO. 1 CHEMOTHERAPY computer programmed to perform image segmentation, feature evaluation, decision making and data analysis have been used to

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1 CHEMOTHERAPY JAN.1994 Prospects for Monitoring Infectious Diseases Using Computor-Assisted Microscopic Image Analysis Prof. Elliot Goldstein School of Medicine Department of Internal Medicine Division of Infectious Diseases Kansas University School of Medicine Advances in computerized microscopy have resulted in computer-assisted microscopic image analysis systems (CAMIAS) that rapidly and precisely measure various aspects of cellular morphology and physiology. These systems which are composed of a microscope and attached video camera for image acquisition, an image processor, and a

2 VOL. 42 NO. 1 CHEMOTHERAPY computer programmed to perform image segmentation, feature evaluation, decision making and data analysis have been used to detect viral nucleic acids in in situ hybridization preparations, to detect viral antigens within cells, to measure rates of cellular movement, to measure ph and calcium within phagocytes, and to relate the proliferation of intracellular bacteria to reductions in lysosomal enzymes. Because of the importance of determining the presence of HIV-infected cells in evaluating anitiviral chemotherapy, (CAMIAS) have been used to differentiate HIV-infected from uninfected cells. Experiments performed in cell cultures showed that HIV-infected cells can be distinguished by quantifying differences in staining intensity following in situ hybridization reactions with chromogenic signals. The CAMIAS determined the percentage of cells with 500 or more HIV-DNA and HIV-RNA copies within 30 minutes. Similar studies showed enhanced sensitivity when fluorescence was used to detect HIV infected cells. Recent advances in high resolution fluorescent in situ hybridization methodology now permit the detection of a single HIV-genome in a lymphocyte. The clinical use of this sophisticated assay in monitoring HI infections awaits the improvement of currently available instrumentation to allow the rapid enumeration of cells containing these low levels of fluorescence. CAMIAS are also able to enumerate lymphocytes containing HIV-p 24 antigen using immunocytochemical reactions with murine monoclonal antibodies and a peroxidase or fluorescent signaling system. As with HIV nucleic acids, monitoring of cells with HIV-p 24 antigen may aid in assessing effectiveness of therapy. CAMIAS are well suited to measure polymorphonuclear leukocyte (PMN) chemotaxis as they permit continuous measurement of as many as 50 moving PMNs in a microscopic field or continuous measurement of cells moving through filter type chemotactic chambers. Such studies have shown that PMNs from uninfected patients with insulin requiring diabetes have normal rates and responses to F-met-leu-phe during periods of hyperglycemia and that PMNs from patients with severe burns the have reduced motility and responses to F-met-leuphe stimulation. These techniques should be useful in future studies of PMN chemotaxis in infections. CAMIAS have also been used to study the pathophysiology of infection. Techniques employing ph sensitive fluorescent probes have provided important information of the pathogenesis of infections caused by intracellular pathogens. These investigations have shown that Toxoplasma gondii, Legionella pneumophila and virulent strains of Nocardia asteroides cause disease in part by inhibiting phagosomal acidification. Additional studies have shown that calcium is a mediator of cellular locomotion and that intracellular bacterial proliferation reduces lysosomal enzyme concentrations. Thus, although advancement has been slow, future fulfillment to the promise of CAMIAS to provide detailed information of importance in infectious disease can be envisioned in coming years.

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CHEMOTHERAPY JAN, 1994 4) Kohno K, Kuwano M: Is human multidrug resistance 1 a stress-induced SOS gene?

5 CHEMOTHERAPY JAN, ) Kohno K, Kuwano M: Is human multidrug resistance 1 a stress-induced SOS gene? The Mechanism and New Approach on Drug-resistance of Cancer Cells (ed, by Miyazaki T, Takaku F, Sakurada K), Elsevier, Netherlands, p.21-27, ) Takano H, Kohno K, Matsuo K, Matsuda, T, Kuwano M: DNA topoisomerases targeting

6 VOL. 42 NO. 1 CHEMOTHERAPY antitumor agents and drug resistance. Anti-Cancer Drugs (review):3: 323 `330, ) Kohno K, Takano H, Matsuo K, Kiue A, Sato S, Uchiumi T, Miyazaki M, Tanimura H, Sato S, Kuwano M: Drug resistance to DNA topoisomerase 11 targeting agents and development of the reversal agents. Molecular Biology of DNA Topoisomerases ed, by Andoh T, Ikeda H, Oguro M, CRC Press, pp , 1992

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21 CHEMOTHERAPY JAN Grouth Curve of T N F Gene Transduced Meth-A Subclones Inoculated Subcutaneously into Syngenic Mice.

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Fig. 1 Chemical structure of DL-8280

Fig. 1 Chemical structure of DL-8280 Fig. 2 Susceptibility of cl in ical isolates to DL4280 Fig. 5 Susceptibility of clinical isolates to DL-8280 Fig. 3 Susceptibility of clinical isolates to DL-8280 Fig.