Russell A. Faust

Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation

We hypothesized that malignant transformation of normal mucosa of the upper aerodigestive tract to squamous cell carcinoma of the head and neck (SCCHN) might be associated with altered CK2 activity in the chromatin compartment of these tumors. We measured CK2 activity in the cytosol and chromatin of 7 surgical specimens of SCCHN, and 5 specimens of normal oropharyngeal mucosa from non-smokers/non-drinkers. CK2 activity in SCCHN tumors was significantly elevated in both the nuclear chromatin (P < 0.0005) and cytosolic (P <0.04) compartments relative to normal mucosa. These data suggest that activation of dysregulation of the chromatin-associated CK2 signal may play a role in the pathobiology od SCCHN.

Antisense oligonucleotides against protein kinase CK2‐α inhibit growth of squamous cell carcinoma of the head and neck in vitro

Human squamous cell carcinomas of the head and neck (SCCHN) overexpress the protein kinase CK2, and elevated CK2 activity correlates with aggressive tumor behavior and poor clinical outcome. We therefore investigated whether interference with CK2 expression would inhibit SCCHN cell growth in vitro.

Subcellular immunolocalization of protein kinase CK2 in normal and carcinoma cells.

CK2 is a messenger-independent protein serine/threonine kinase that has been implicated in cell growth and proliferation. Our recent analysis of squamous cell carcinomas of the head and neck (SCCHN) revealed a significant elevation in CK2 activity in these tumor cells relative to normal mucosa of the upper aerodigestive tract and suggested a correlation with aggressive tumor behavior and poor clinical outcome. In order to further define the distribution of CK2 in these tissues, we have examined the immunohistochemical staining pattern of surgical specimens of both SCCHN tumors and normal upper aerodigestive tract mucosa using a monoclonal antibody directed against the catalytic subunit CK2-alpha of the kinase, and have compared these data with the subcellular distribution of CK2 activity in these same tissues. These measurements showed that CK2 is predominantly localized to the nuclei of the tumor cells, which agreed closely with the immunohistochemical staining pattern of CK2-alpha in tumor cells. The chiefly nuclear distribution of CK2-alpha immunostaining found consistently in SCCHN tumor cells and tumor-infiltrating lymphocytes contrasted with a relatively more predominant cytosolic staining pattern exhibited by various cellular constituents of normal oropharyngeal mucosa. The immunostaining pattern of CK2-alpha revealed that staining was observed in the cells stained for the proliferation-marker Ki-67; however, strong distinct immunostaining for CK2-alpha was also observed in large numbers of other cells in these same tumors, suggesting that CK2 elevation in these tumors is not a reflection of proliferative activity alone, but may also relate to the pathobiological behavior of the tumor.

Significance of protein kinase CK2 nuclear signaling in neoplasia.

Many stimuli play a role in influencing the structure and function of chromatin and nuclear matrix through post‐translational modifications of the component proteins in these dynamic structures. We propose that the protein serine/threonine kinase CK2 (formerly casein kinase II) is one such agent that is involved in signal transduction in the nuclear matrix and chromatin in response to a variety of stimuli. Protein kinase CK2 appears to undergo rapid modulations in its association with nuclear matrix and nucleosomes in response to mitogenic signals and is involved in the phosphorylation of a variety of intrinsic proteins in these structures depending on the state of genomic activity. In addition, its association or loss from the nuclear matrix may also influence the apoptotic activity in the cell. CK2 has been found to be dysregulated in virtually all the neoplasias examined and nuclear association appears to be an important facet of its expression in tumor cells. We hypothesize that CK2 provides a functional paradigm linking the nuclear matrix and chromatin structures. Identification of precise loci of action of CK2 in these structures and how they influence the morphological appearance of the nucleus under normal and abnormal growth conditions would be an important future direction of investigation. J. Cell. Biochem. Suppl. 35:130–135, 2000. Published 2001 Wiley‐Liss, Inc.

Outpatient biopsies of the palatine tonsil: Access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers

OBJECTIVES Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patients lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN A pilot cohort study was performed. PATIENTS We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.

Association of protein kinase CK2 with nuclear matrix: Influence of method of preparation of nuclear matrix

Nuclear matrix (NM) plays roles of fundamental structural and functional significance as the site of replication, transcription, and RNA processing and transport, acting as an anchor or attachment site for a variety of enzymes and other proteins involved in these activities. We have previously documented that protein kinase CK2 translocates from the cytosol to the nucleus, where it associates preferentially with chromatin and NM, in response to certain growth stimuli. Considering that characteristics of the isolated NM can depend on the procedure employed for its isolation, we compared three standard methods for NM preparation to confirm the association of intrinsic CK2 with this structure. Our data suggest that the method used for isolating the NM can quantitatively influence the measurable NM‐associated CK2. However, all three methods employed yielded qualitatively similar results with respect to the stimulus‐mediated modulation of NM‐associated CK2, thus further supporting the notion that NM is an important site for physiologically relevant functions of CK2. In addition, core filaments and cytoskeleton that were isolated by two of the preparative methods had a small but significant level of associated CK2 activity. J. Cell. Biochem. 64:499–504.

Modulation of nuclear matrix protein phosphorylation by histones: Possible involvement of NM-associated protein kinase CK2 activity †

Nuclear matrix (NM), a proteinaceous network of filaments, dictates nuclear morphology and the structure/function of DNA. Phosphorylation of NM proteins is a potential signal for regulating matrix functions. Histones also are intimately involved in DNA structure and transcription. Here, we report that various histones enhanced 32P incorporation into certain NM proteins. Modulation of NM protein phosphorylation by histones is mediated through regulation of protein kinase CK2, a messenger‐independent serine/threonine kinase, which is significantly associated with the NM. The stimulatory effect of histones was mitigated by prior incubation of histones with DNA in the reaction. Phosphorylation of NM proteins was extensively reduced when an excess of the CK2‐specific peptide substrate was included in the phosphorylation reaction as a competitor. Also, enhancement in the NM‐associated CK2 activity by histones was blocked by inhibitors of CK2. Histone H1 effect appeared to be mediated mainly by charge effect since a stretch of polylysine induced a similar effect. Various histones also differentially affected the autophosphorylation of NM‐associated CK2 subunits. This may contribute to the observed effects of histones on the NM, resulting in an enhancement and differential pattern of NM protein phosphorylation. Such a regional modification of NM protein phosphorylation might influence the nuclear functions that require histone displacement, namely, replication and transcription. J. Cell. Biochem. 72:242–250, 1999.

11:00 AM: Dynamic, Real-Time Magnetic Resonance Imaging: Evaluation of the Larynx and Tracheal Airway

Objective: Young or early career professional singers have a relatively high incidence of voice disorders when compared with more mature or experienced singers. If the factors that predispose these singers to vocal disorders could be identified and a high-risk registry developed, then an interventional program could be designed to prevent or moderate the occurrence of these disorders. Methods: For 3 years, a cohort of nearly 100 singers at a major national commercial theme park were interviewed and followed up. The initial evaluation included a detailed vocal and medical history, singing audition, and interview by the laryngologist. Results: Fifteen percent of the singers were initially classified as high risk, 18% as moderate risk, and the rest as low to no risk on the basis of the results of the evaluation. In year 1, no preventive program was developed and the singers were seen when ill. Ninety-two percent of the high-risk singers and 78% of the moderate-risk singers presented with vocal disorders; only 8% of the low-risk singers were seen. In years 2 and 3, a preventive vocal program was initiated using didactic education sessions on diet, vocal pathophysiology, smoking, allergy care, vocal abuse and misuse disorders, and lifestyle. A personalized program was developed for each singer in the high-risk profile. Medical surveillance by the laryngologist was performed on site at the park. After initiation of the preventive program, the overall rate of visits to the laryngologist decreased by 85% in the highand moderate-risk groups. Conclusion: Medical preventive and surveillance programs can be used to identify and follow up on highand moderate-risk singers. Such a program could also be used in secondary schools and university vocal arts programs.

Molecular Biology 101 for the Practitioner

Educational objective: To acquire a basic understanding of the concepts and research methodology used in molecular otolaryngology.