Alanine aminotransferase levels are not significantly elevated in patients with HIV/HBV co-infection and lamivudine resistance.

In hepatitis B virus (HBV) monoinfection, alanine aminotransferase (ALT) levels are linearly correlated with HBV DNA levels and lamivudine resistance. In human immunodeficiency virus (HIV)/HBV co-infection, little is known about the association between ALT, HBV DNA, and lamivudine resistance. We assessed HBV DNA, lamivudine resistance and ALT levels in 45 time points in 11 patients with HIV/HBV co-infection during lamivudine-containing antiretroviral therapy. High HBV DNA levels (>10(6) copies/mL) and lamivudine resistance developed in 45% and 91% of patients, respectively. However, ALT levels were not elevated in the setting of high HBV DNA levels (mean ALT, 48 IU/mL) or lamivudine resistance (mean ALT, 44 IU/mL). HBV viraemia and lamivudine resistance during extended lamivudine-containing antiretroviral therapy are common in HIV/HBV co-infection, occurring in the absence of significant ALT elevations. In HIV/HBV co-infection, measurement of HBV DNA and HBV resistance mutations may identify HBV virological failure before biochemical changes and should be routinely used in the management of HIV/HBV co-infection.

Quantitative microscopy: I. A computer-assisted approach to the study of polymorphonuclear leukocyte (PMN) chemotaxis.

A computer-assisted approach has been designed to analyze and quantitate polymorphonuclear leukocyte (PMN) chemotaxis. This approach involves a rapid, objective, and semiautomated (user-directed) image-analysis system that is video- and microscope-based. The entire system consists of a microvideo set-up that is put on line with a Digital DEC-LSI-11/73 microcomputer, interfaced with a Datacube analog-digital/digital-analog converter. Video signals of PMN movement are digitized by the system at a resolution of 240 pixels vertically by 320 pixels horizontally (at 256 gray levels) and stored in a 76,800-byte frame buffer. The digitized data are stored for later use or utilized immediately for image segmentation, image display, movement, and morphometric computations for each PMN in a maximum phase field (at 645 X high dry) of 50 PMNs at 10-second intervals. The digitized data are used for computation of cell perimeter, surface area, optical density, contour-ratio, position, speed, and direction of locomotion with the utilization of micro-image-analysis programs written in FORTRAN and MACRO assembly language, with the computer operating under RT-11/TSX+. The reliability, objectivity, and reproducibility of measurements made with this quantitative approach have been tested by comparing with manual-tracing measurements of PMN movement. A correlation factor of 0.99 has been obtained. However, the quantitative-microscopic approach is much faster, more objective, less tedious, and much easier to operate than the conventional manual-tracing method.

Reactivation of tritonated models of human polymorphonuclear leukocytes (PMNs): a computer-assisted analysis.

The orientation (chemotaxis) and locomotion (chemokinesis) of human polymorphonuclear leukocytes (PMNs) are generated by an internal movement mechanism that involves active cytoplasmic movement; they are influenced by external environmental and ionic conditions. We have studied the degree to which the orientation and movement mechanisms of PMNs are self-contained within the cell and the degree to which they are under membrane control. PMNs were partially and selectively demembranated by treatment with the non-ionic detergent, octyl-phenoxyl-polyethoxyethanol (commercially known as Triton X-100) under controlled conditions. The tritonated PMNs (referred to in the literature as models) were non-motile and non-locomotory. Addition of ATP/Mg++ with a trace amount of Ca++ to the medium was followed by reactivation of the tritonated PMN models to move again as motile cells. Although these reactivated PMN models actively locomoted, they could no longer orient to chemoattractants. Thus, the reactivation process restored the physical self-contained movement parameters but could not reestablish the orientation capacity (chemotactic responsiveness) that was characteristic of live PMNs. The demembranation process apparently destroyed the chemotactic receptors and/or eradicated the coordination function of the membrane. Videotapes of normal (control) as well as reactivated PMN movement were analyzed for movement characteristics. These characteristics were objectively analyzed with a newly designed computer-assisted micro-image-processing technique whereby the videotapes were digitized and quantified and the actual PMN movement printed out in computer-graphics and tracings (Freeman codes) for confirmation of orientation and movement arising as a result of reactivation.

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines.

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.

HIV-1 proviral copy number in blood mononuclear cells from AIDS patients on zidovudine therapy.

The effect of treatment with 400-1,200 mg/day of zidovudine (ZDV) on HIV DNA concentrations in patient peripheral blood mononuclear cells (PBMCs) was studied in six patients during a 5- to 14-month period of therapy. HIV DNA was measured in PBMCs at intervals using a recently developed quantitative polymerase chain reaction assay. The amount of HIV DNA ranged from 2,000 to 40,000 copies of provirus per microgram of cellular DNA. The HIV provirus copy number showed little change with time in five patients, and increased and then remained constant in one patient. Thus, prolonged treatment with ZDV does not decrease the levels of HIV DNA in PBMCs.

Antiretroviral therapy is associated with a decrease in unintegrated HIV-1 DNA in pediatric patients.

Good markers for monitoring the efficacy of antiretroviral therapy in children do not currently exist. This study examined the effect of antiretroviral therapy on human immunodeficiency virus (HIV-1) unintegrated DNA (uDNA), integrated DNA (iDNA), percent uDNA, immune complex dissociated (ICD) p24 antigenemia, and plasma viral titer. Seven children were followed at therapy initiation and at approximately 3- and 10-month intervals. HIV-1 uDNA was detected in all children prior to start of therapy (average percent uDNA, 43%). At 3 months, the percent HIV uDNA decreased in all patients to an average of 18% (p = 0.01) and at 10 months decreased to an average of 1%. In contrast, the amount of HIV iDNA was relatively constant after initiation of therapy. ICD HIV p24 antigen was detected in all patients prior to therapy (average, 538 pg/ml). Over the study period, the ICD p24 antigen level decreased in three patients and remained relatively unchanged in four patients. Plasma cultures of HIV-1 were positive in only one of the seven patients prior to therapy. Among the methods evaluated, measurement of uDNA was the only parameter which reliable decreased after initiation of nucleoside therapy.

Decreases in unintegrated HIV DNA are associated with antiretroviral therapy in AIDS patients.

Better markers are needed to monitor the efficacy of antiretroviral drugs in persons infected with human immunodeficiency virus (HIV). We investigated the effects of zidovudine (ZDV) and dideoxycytidine (ddC) on the presence of unintegrated HIV-1 DNA in peripheral blood mononuclear cells (PBMCs) from AIDS patients. DNA was extracted from PBMCs and separated into low molecular weight (unintegrated) and high molecular weight (integrated) chromosomal fractions. These DNA fractions were then amplified by a quantitative polymerase chain reaction (PCR) and the amount and percentage of unintegrated HIV DNA were determined. Very high levels of unintegrated HIV DNA were found in AIDS patients not receiving treatment with ZDV or ddC (median = 95% unintegrated HIV DNA). In contrast, most patients who had received 4 or more weeks of antiretroviral therapy had lower levels of unintegrated HIV DNA (median = 30% unintegrated HIV DNA for patients receiving ZDV). Paired samples taken from five patients before and after therapy showed a striking reduction in the percentage of unintegrated HIV DNA. The decrease in the proportion of unintegrated HIV DNA in AIDS patients was due to both a reduction in the copy number of unintegrated HIV DNA and an increase in the copy number of integrated HIV DNA. Thus, measurements of unintegrated and integrated HIV DNA may be useful in providing objective assessments of the effectiveness of antiretroviral therapies.

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry.

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.

In situ detection of human immunodeficiency virus (HIV) nucleic acid in H9 cells using nonradioactive DNA probes and an image cytophotometry system.

Rapid and sensitive nonradioactive methods to detect human immunodeficiency virus (HIV)-infected cells are needed in clinical medicine. We developed an in situ hybridization test using 2-acetylaminofluorene (AAF)-labeled HIV DNA as a hybridization probe. Hybridized probe was detected using rabbit anti-AAF antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit, and the bromochloroindolyl phosphate-nitroblue tetrazolium reaction. An image cytophotometry system was used to quantitate the percentage of HIV-infected cells. These methods were used to determine the percentage of H9 cells infected with HIV. HIV was detected in 0% of cells on day 1 post infection, 7% on day 4, 41% on day 8, and 5% on day 15. These results paralleled those of the reverse transcriptase assay and an antigen capture ELISA assay for HIV antigen. Thus the AAF modified HIV DNA probe detected HIV nucleic acid in infected H9 cells and the image cytophotometry system improved the sensitivity and objectivity of detection.

Quantitation of unintegrated HIV-1 DNA in asymptomatic patients in the presence or absence of antiretroviral therapy.

The objective of this work was to determine the amount of unintegrated human immunodeficiency virus (HIV) DNA (HIV uDNA) in asymptomatic individuals in the presence or absence of antiretroviral therapy. Twenty-one healthy seropositive individuals with no history of any opportunistic infection or previous use of nucleoside antiretrovirals, and 9 similarly asymptomatic individuals who had initiated nucleoside antiretroviral therapy within the last 24 months were studied. All patients had CD4 lymphocyte counts above 400/microliters. All subjects administered antiretrovirals received 400-600 mg of zidovudine daily for 2-24 months. Two individuals additionally received 400 mg of dideoxyinosine (ddI) daily for 4 and 5 months. Patient peripheral blood mononuclear cells (PBMCs) were examined for integrated and unintegrated HIV DNA by a quantitative PCR assay. In addition, CD4 counts were measured, and free and immune complex dissociated p24 antigen was detected in plasma by ELISA. The mean percentage of HIV uDNA in asymptomatic individuals not on therapy was 59%, with 95% confidence limits from 50 to 69%. In contrast, patients on therapy had a mean of only 13% HIV uDNA, with confidence limits from 2 to 25% (p < 0.001). These findings indicate that a significant amount of HIV DNA in infected, healthy patients not on therapy is in the unintegrated form, and that the amount of HIV uDNA in asymptomatic patients on nucleoside therapy is much less. The amount of HIV uDNA in PBMCs deserves further study as a new marker of the efficacy of antiretroviral therapy.

Comparison of HIV type 1 RNA plasma viremia, p24 antigenemia, and unintegrated DNA as viral load markers in pediatric patients.

Better surrogate markers need to be developed to evaluate therapy in HIV-infected children. This study evaluated plasma RNA, immune complex-dissociated p24 antigenemia, and unintegrated DNA (uDNA) in HIV-infected pediatric patients. Ten children were followed from initiation of nucleoside antiretroviral therapy at intervals up to 24 months. Prior to initiation of therapy, HIV RNA was detected in 10 of 10 patients (median, 76,000 Eq/ml), p24 antigen was detected in 8 of 10 patients (median, 193 pg/ml), and uDNA was detected in 6 of 7 patients (median, 10% uDNA). After 12 months the RNA decreased in all patients and became undetectable in six. In contrast, p24 antigenemia decreased in 6 of 10 patients, remained undetectable in 1, and increased in 3. HIV uDNA decreased in six of six patients and became undetectable in three. There was no overall change in CD4 cell count. Plasma RNA and uDNA levels are both sensitive markers of nucleoside therapy in children; however, they do not covary strongly.

Monocyte function in rhesus monkeys with simian acquired immune deficiency syndrome.

Monocyte function in rhesus monkeys with simian acquired immune deficiency syndrome (SAIDS) was compared with that in age-matched normal juvenile rhesus monkeys. The functional tests were 1) chemotaxis, 2) phagocytosis of opsonized Candida albicans, 3) killing and/or growth inhibition of Candida albicans, 4) generation of respiratory burst, and 5) monocyte-derived macrophage response (morphology and/or respiratory burst) to stimulating agents such as lymphokines, gamma interferon, endotoxin, and phorbol myristate acetate. The monkeys tested had either clinical SAIDS (alive with lymphadenopathy, splenomegaly, and lymphopenia or neutropenia) or had terminal SAIDS (moribund due to the disease). Responses of monocytes from 14 monkeys with clinical SAIDS were indistinguishable from those of 9 normal juvenile rhesus monkeys, whereas monocytes from 3 monkeys with terminal SAIDS had enhanced phagocytosis and respiratory burst capacity. Chemotaxis, candidacidal/stasis activity, and response to stimulating agents were normal in these terminal cases. Plasma from the SAIDS monkeys was as capable of opsonizing yeasts and of being able to generate chemotactic factors by endotoxin as was control plasma. SAIDS retrovirus (SRV) was detected by co-cultivation of pure monocyte-derived macrophage cultures with Raji cells, an indicator cell line which forms syncytia in the presence of SRV. Four terminal SAIDS cases and one late-stage clinical SAIDS case were virus-positive when the number of macrophages in the cultures ranged from less than 50 to about 500. Terminal SAIDS monocyte-derived macrophages in culture as long as 17 days produced SRV. These data show that in monkeys with SAIDS the major effector functions of monocytes and macrophages involved in host defense are intact (even up until death). Additionally, some of the monocytes are productively infected, and these infected monocytes are viable and adherent in culture.

Laboratory tests used in diagnosis and treatment of AIDS.

Detailed knowledge of the structure of the human immunodeficiency virus (HIV) triggered a rapid development of methods for diagnosing the infection. The enzyme-linked immunoadsorbent assay (ELISA) determining the presence of antibodies to the protein components of the virus in toto is highly sensitive and provides thus the basic screening approach. It is however somewhat less specific and therefore all positive results are verified by the Western blot method which detects individual HIV proteins and possesses a 99.9% specificity. In sporadic cases the latent period between HIV infection and the possibility to establish antibody response may extend to six months and even longer. The polymerase chain reaction (PCR) revealing the presence of DNA HIV in infected cells is suitable for detecting these seronegative patients. It is also the method of choice in diagnosing HIV infection in children of infected mothers since HIV antibodies are in newborns mostly of maternal origin. HIV antibodies, particularly the antigen p 24, can be quantified in serum by using ELISA. As their amounts correlate with the severity of the disease determination of antigen p 24 concentration is an indicator of the therapeutic efficacy of preparations such as AZT. The recently developed quantitative PCR is a further potentially valuable method for assessing the therapeutic effect since treatment should reduce the amount of cellular HIV DNA. With the exception of testing the sensitivity of HIV strains to antiretrovirus preparations, HIV cultures are rarely set up since they are technically demanding and involve a considerable risk.

Unusual sites of acute osteomyelitis in childhood.

In children osteomyelitis is common in the long hours of femur, tibia and humerus. This study reports 16 children, aged 2-13 years, with osteomyelitis at unusual sites: in the bones of the thoracic cage including three involving the clavicle; in the spine, foot and elsewhere. In one case, multifocal involvement of the vertebral body and the knee occurred. In two large series reported previously, the incidence of osteomyelitis was 1-3% in the clavicle, 3-8% in the calcaneus and less than 1% in the ribs. Four out of 16 cases (two involving clavicles, one rib and one with multifocal sites of the lesion) required open biopsies and histological examination to achieve the final diagnosis of osteomyelitis; in three of these patients the causative agent was not identified on culture. Staphylococcus aureus was the infective organism in 50% of cases where cultures were obtained. In five cases there was no growth on culture and specific search for less common organisms, including mycobacteria tuberculosis (AAFB), proved negative. It is suggested that in such situations diagnostic problems may present as the clinical and radiological findings may not be specific or conclusive. In such cases early biopsy is mandatory.

Applications of computerized microscopic image analysis in infectious diseases.

Advances in computerized microscopy have resulted in image analysis systems that rapidly and precisely measure various aspects of cellular morphology and physiology. These systems-composed of a microscope and attached photomultiplier tube or camera, an image processor, and a computer-have been used to measure lysosomal enzymes, pH, and calcium within phagocytes; to detect viral nucleic acids in in situ hybridization preparations; and to quantitate rates of cellular movement. These experiments have shown that (1) the intracellular proliferation of virulent microorganisms is associated with reductions in acid phosphatase, beta-glucuronidase, and lysozyme activity; (2) virulent Toxoplasma gondii, Legionella pneumophila, and Nocardia asteroides inhibit phagosomal acidification; and (3) changes in intracellular calcium movement affect phagocytic function. These methods have also been used to detect the AIDS virus within cultured lymphocytes and to measure cellular chemotaxis and chemokinesis. Further advances in technology should produce improved microscopic image analysis systems with wider applications for the investigation of infectious diseases.

Human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa.

Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function.

Acidification of phagosomes in murine macrophages: blockage by Nocardia asteroides.

Most strains of Nocardia asteroides are susceptible to the detrimental effects of pH 5 when grown in buffered brain-heart infusion broth. Preventing phagosomal acidification may be a mechanism by which this organism survives the microbicidal activity of macrophages. Fluorescein isothiocyanate was conjugated to the surface of Nocardia and Saccharomyces to form pH-sensitive fluorescent probes. The fluorescent emission, and thus the pH, of this probe was quantitated within individual phagosomes by using a computerized cytospectrophotometer. When either live or dead cells of virulent N. asteroides strain GUH-2 were ingested, the phagosomal pH remained above pH 7 for 2 hr. A nonpathogenic soil isolate, N. asteroides strain 19247, only partially blocked acidification. In contrast, when Saccharomyces was used as a control for normal response, the pH decreased to approximately pH 5. Therefore, virulent N. asteroides blocks phagosomal acidification. Because killed Nocardia act in the same manner, this inhibition of acidification appears to be associated with cellular components. This capacity to prevent phagosomal acidification may be prerequisite to the survival of intracellular pathogens.

Modulation of lysosomal protease-esterase and lysozyme in Kupffer cells and peritoneal macrophages infected with Nocardia asteroides.

Virulent Nocardia asteroides reduces lysosomal acid phosphatase activity in murine macrophages. A computer-assisted imaging photometry system was used to quantitate lysozyme and nonspecific esterase-neutral protease levels within individual macrophages following ingestion of nocardiae. In contrast to acid phosphatase, lysozyme and esterase-neutral protease activity was either unchanged or increased following infection by increasing numbers of nocardial cells.

A study of HIV RNA viral load in AIDS patients with bacterial pneumonia.

We examined the effect of bacterial pneumonia on the magnitude of circulating plasma HIV RNA in HIV-infected patients. Serum samples from 13 adult HIV-infected patients (median CD4 count = 83 cells/microl) were assayed for HIV RNA using the reverse transcriptase polymerase chain reaction assay (a) before bacterial pneumonia, (b) during the acute phase, and (c) after the recovery from the disease. Patients remained on constant antiretroviral therapy: HIV RNA was detected in all samples tested. The medians before, during, and after bacterial pneumonia were 60,000 copies per ml, 245,000 copies per ml, and 84,000 copies per ml, respectively. All 13 patients had increased HIV RNA levels on developing pneumonia. There was a decline in the level of HIV RNA with recovery from pneumonia in 12 of 13 patients. The difference between the HIV RNA levels before and after pneumonia was not significant, nor was there significant difference in the CD4 counts before and after pneumonia. In conclusion, bacterial pneumonia is associated with a consistent, transient increase in HIV RNA of variable magnitude in AIDS patients. Interpretation of HIV RNA changes for clinical management of AIDS patients must take into account this reversible elevation during infections.