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Biomedical subjects

C J Miller

Publications and source records attributed to C J Miller.

At least 109 records · Page 6Linked to original sources
PubMed1994

Extensively methylated myosin subfragment-1: examination of local structure, interactions with nucleotides and actin, and ligand-induced conformational changes.

The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.

Actins↗
PubMed1994

Engineering poliovirus as a vaccine vector for the expression of diverse antigens.

As a step toward developing poliovirus as a vaccine vector, poliovirus recombinants were constructed by fusing exogenous peptides (up to 400 amino acids) and an artificial cleavage site for viral protease 3Cpro to the amino terminus of the viral polyprotein. Viral replication proceeded normally. An extended polyprotein was produced in infected cells and proteolytically processed into the complete array of viral proteins plus the foreign peptide, which was excluded from mature virions. The recombinants retained exogenous sequences through successive rounds of replication in culture and in vivo. Infection of animals with recombinants elicited a humoral immune response to the foreign peptides.

Amino Acid Sequence↗
PubMed1994

Protein phosphatase activity against protein kinase C-phosphorylated substrates in human placenta.

The presence of endogenous modulators of protein kinase C (PKC) in human placenta has not been reported. The specific activity of PKC in human placental cytosol was 20.52 +/- 1.8 pmol/min x mg protein. Partial purification of placental cytosol on diethylaminoethyl cellulose (DEAE) resulted in recovery of 145 per cent of original enzyme activity. Placental cytosol mixed with a control preparation of PKC significantly inhibited the control enzyme activity (control 42.42 +/- 2.8 pmol/min; control+placental cytosol 27.44 +/- 2.8 pmol/min, P < 0.05). The PKC-inhibitory activity was abolished by the addition of phosphatase inhibitors calyculin A (0.09 nM), microcystin LR (0.8 nM), and okadaic acid (0.4 nM). Protein substrates phosphorylated by PKC were rapidly dephosphorylated upon the addition of placental cytosol; this dephosphorylation was prevented by the presence of calyculin A and was removed by fractionation of placental cytosol on DEAE. Protein but not peptide substrate supported both the PKC-inhibitory activity and the dephosphorylation of PKC-phosphorylated substrates. The placental serine-threonine protein phosphatase was active against phosphorylase a, but not against substrate phosphorylated by cAMP-dependent protein kinase. These data indicate that the human placenta contains an endogenous inhibitor of PKC which interacts with substrate rather than with the PKC and that the inhibitor is a protein phosphatase.

Calcium↗
PubMed1994

The common mucosal immune system: from basic principles to enteric vaccines with relevance for the female reproductive tract.

The realization that induction of immune responses at mucosal surfaces may prevent colonization, invasion or dissemination of pathogenic microorganisms has spurred intensive efforts to develop vaccines which elicit effective mucosal immunity. In this paper, recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses. Oral administration of cholera toxin alone or with a co-administered protein vaccine tetanus toxoid induces a strong T helper type 2 (TH2) cell response in both Peyer's patches and spleen. Both serum IgG and secretory IgA antibodies specific for cholera toxin or for the co-administered protein tetanus toxoid were induced. When administered parentally, however, no mucosal antibody responses were evident and a mixed TH1- and TH2-type CD4+ T cell response was noted in the spleen. Various vectors are being employed in an effort not only to induce mucosal immune responses but also to direct the response to a TH1-type response, thought to promote strong cell-mediated immune responses, or to a TH2-type response for maximum B cell antibody responses. The ability to manipulate the TH cell responses may provide a more rational approach for the design of vaccines. Although lymphoid tissues of the female reproductive tract differ from that of the gut, many of the strategies and evolving principles may be directly applicable to the development of vaccines designed to prevent sexually transmitted diseases.

Administration, Oral↗
PubMed1994

Primary acute simian immunodeficiency virus infection of intestinal lymphoid tissue is associated with gastrointestinal dysfunction.

Human immunodeficiency virus (HIV)-associated intestinal abnormalities can occur before immunodeficiency or infection with opportunistic enteropathogens. Rhesus macaques infected with simian immunodeficiency virus (SIV) develop an AIDS-like illness that frequently includes enteropathy. The development of enteropathy and its association with SIV infection in the intestinal tract was examined. By 1 week after infection, SIV-infected macrophages and T lymphocytes were detected in gut-associated lymphoid tissue. In contrast to findings in the asymptomatic stage, SIV-infected macrophages were numerous in primary and terminal stages of infection. An acute enteropathy syndrome was observed in the primary acute stage of infection. Functional abnormalities of absorptive epithelium, indicated by D-xylose malabsorption and decreased sucrase activity, occurred before the onset of diarrhea or opportunistic enteric infections. These findings indicate that macrophages and T cells in the intestinal tract are early targets of SIV infection and may play a critical role in the development of SIV-associated intestinal dysfunction.

Acute Disease↗
PubMed1994

Development of malabsorption and nutritional complications in simian immunodeficiency virus-infected rhesus macaques.

OBJECTIVE: To assess the development and cause of malabsorption in rhesus macaques following experimental simian immunodeficiency virus (SIV) infection and to evaluate its impact on nutritional status. DESIGN: Clinical malabsorption tests and serial jejunal aspirates and biopsies were obtained from nine SIV-infected and three uninfected animals prior to infection and at 1, 2, 4, 8, 12, 24, 40 and 52 weeks postinoculation. METHODS: Malabsorption was measured by sucrose breath hydrogen (H2) analysis and blood assay of D-xylose. Digestive enzyme activity was determined in jejunal mucosal homogenates. Bacterial and protozoal flora were determined in jejunal aspirates. Nutritional assessment was evaluated using specific blood micronutrient values. Cellular targets of SIV in the jejunum were determined by combined in situ hybridization and immunohistochemistry. RESULTS: Eight out of nine SIV-infected monkeys, including asymptomatic animals, exhibited malabsorption by either increased breath H2 and/or decreased blood D-xylose. All animals that died of AIDS had diarrhea, D-xylose malabsorption and decreased sucrase activity. Significant changes in nutritional status were associated with malabsorption. Bacterial overgrowth was not considered to be a cause of malabsorption. Histopathological biopsy findings included dilated villus lacteals, excessive cellular debris, lymphoplasmocytic infiltrates and cytoplasmic vacuoles in crypt epithelial cells. SIV-infected T cells and macrophages were detected as early as 1 week postinoculation. CONCLUSIONS: SIV-associated malabsorption can occur prior to clinical complications of disease. Severe intestinal complications are associated with malabsorption and malnutrition in the terminal stages of AIDS. The high proportion of macaques experiencing malabsorption without detectable pathogens, suggests an enteropathogenic role for SIV.

Animals↗
PubMed1994

Development of a physical performance and mobility examination.

OBJECTIVE: To develop and validate the Physical Performance and Mobility Examination (PPME), an observer-administered, performance-based instrument assessing 6 domains of physical functioning and mobility for hospitalized elderly. DESIGN: Development of a pass-fail and 3-level scoring system and training manuals for the PPME instrument for use in both clinical and research settings. Two patient samples were used to assess construct validity and interrater reliability of the PPME. A third sample was selected to assess the test-retest reliability of the instrument. SETTING/PATIENTS: (1) 146 subjects > or = 65 years of age with impaired mobility admitted to Medical Units of Stanford University Hospital. (2) 352 subjects > or = 65 admitted to acute Medical and Surgical Services of the Palo Alto VA Medical Center. Patient samples were obtained during hospitalization and followed until 3 months post-discharge. To study test-retest reliability, 50 additional patients, whose clinical condition was stable, were selected from both settings. METHODS: An expert panel selected 6 mobility tasks integral to daily life: bed mobility, transfer skills, multiple stands from chair, standing balance, step-up, and ambulation. Tasks were piloted with frail hospitalized subjects for appropriateness and safety. Test-retest and interrater reliability and construct validity were evaluated. Construct validity was tested using the Folstein Mini-Mental State Examination, Activities of Daily Living (ADL), Instrumental Activities of Daily Living (IADL), Geriatric Depression Scale, and modified Medical Outcomes Study Measure of Physical Functioning (MOS-PFR). Two scoring schema were developed for each task: (1) dichotomous pass-fail and (2) 3-level high pass, low pass, and fail. A summary scale was developed for each method of scoring. MAIN RESULTS: High interrater reliability and intrarater reliability were demonstrated for individual tasks. The mean percent agreement (interrater) for each pass/fail task ranged from 96 to 100% and from 90 to 100% for the 3 pairs of raters for each task using the 3-level scoring. Kappas for individual pairs of raters ranged from .80 to 1.0 for pass-fail scoring and from .75 to 1.0 for 3-level scoring (all P < 0.01). Intraclass correlation coefficients for 3-level scoring by pairs of raters ranged from .66 to 1.0. For summary scales, the mean intraclass correlation was .99 for both scoring schema. Test-retest reliability for summary scales using kappa coefficients was .99 for both pass-fail and 3-level scoring, and .99 and .98, respectively, using Pearson Product Moment Correlation. Correlations of PPME with other instruments (construct validity) suggest that the PPME adds a unique dimension of mobility beyond that measured by self-reported ADLS and physical functioning, and it is not greatly influenced by mood or mental status (r = 0.70 (ADL), r = 0.43 (IADL), r = 0.36 (MMSE), r = 0.71 (MOS-PFR), r = 0.23 (GDS)). The 3-level summary scale was sensitive to the variability in the patient population and exhibited neither ceiling nor floor effects. CONCLUSIONS: The PPME is a reliable and valid performance-based instrument measuring physical functioning and mobility in hospitalized and frail elderly.

Activities of Daily Living↗
PubMed1994

Intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia.

The simian immunodeficiency virus (SIV)-rhesus macaque model of heterosexual human immunodeficiency virus transmission consists of atraumatic application of cell-free SIVmac onto the intact vaginal mucosa of mature female rhesus macaques. This procedure results in systemic infection, and eventually infected animals develop the clinical signs and pathologic changes of simian AIDS. To achieve 100% transmission with the virus stocks used to date, multiple intravaginal inoculations are required. The current titration study utilized two stocks of SIVmac and demonstrated that a single intravaginal dose of cell-free SIV can reliably produce infection in rhesus macaques. This study also demonstrated that some animals intravaginally inoculated with cell-free SIVmac develop transient viremia characterized by a limited ability to isolate virus from peripheral blood mononuclear cells and lymph node mononuclear cells and no seroconversion to SIV antigen. SIV could be isolated from the peripheral lymph nodes of transiently viremic animals only during periods of viremia and not at times when SIV was not detected in circulating mononuclear cells. Thus, peripheral lymphoid tissues were not reservoirs of infection in the transiently viremic animals. Taken together, these results suggest either that the SIV infection was cleared in the transiently viremic animals or that SIV infection is limited to a compartment of the genital mucosal immune system that cannot be assessed by monitoring SIV infection in peripheral blood mononuclear cells and peripheral lymphoid tissue.

Animals↗
PubMed1994

A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge.

Three infectious, attenuated molecular clones of simian immunodeficiency virus (SIVmac) were tested for viral and host determinants of protective immunity. The viruses differed in degree of virulence from highly attenuated to moderately attenuated to partially attenuated. Levels of immune stimulation and antiviral immunity were measured in rhesus macaques inoculated 2 years previously with these viruses. Monkeys infected with the highly attenuated or moderately attenuated viruses had minimal lymphoid hyperplasia, normal CD4/CD8 ratios, low levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against p55gag (Gag) or gp160env (Env). Monkeys infected with the partially attenuated virus had moderate to marked lymphoid hyperplasia, normal CD4/CD8 ratios, high levels of SIV-specific antibodies, and cytotoxic T-lymphocyte activity against both Gag and Env. After pathogenic virus challenge, monkeys immunized with the partially attenuated virus had 100- to 1,000-fold-lower viral load in peripheral blood mononuclear cells and lymph node mononuclear cells than naive control animals. One of four monkeys immunized with the highly attenuated virus and two of four monkeys immunized with the moderately attenuated virus developed similarly low viral loads after challenge. These three attenuated strains of SIV induced a spectrum of antiviral immunity that was inversely associated with their degree of attenuation. Only the least attenuated virus induced resistance to challenge infection in all immunized monkeys.

Animals↗
PubMed1994

Pathology and localization of simian immunodeficiency virus in the reproductive tract of chronically infected male rhesus macaques.

BACKGROUND: Despite the fact that human immunodeficiency virus (HIV) is transmitted by sexual contact, the biology of this mode of transmission remains largely undefined. HIV is present in semen in both cell-free and cell-associated forms and can be isolated from asymptomatic individuals and AIDS patients. The source of HIV in semen and the target cells for HIV transmission to men are unknown. Epidemiologic studies have shown that in men, the presence of an intact foreskin is associated with increased risk of HIV infection. EXPERIMENTAL DESIGN: The reproductive tracts from 13 chronically simian immunodeficiency virus (SIV)-infected adult male rhesus macaques were examined for this study. Routine histologic examination was undertaken to characterize the reproductive tract pathology. In situ hybridization and immunohistochemistry were used to localize SIV-infected cells and determine the immunophenotype of inflammatory infiltrates. RESULTS: SIV-infected cells (macrophages, T cells) were found at all levels of the reproductive tract and were commonly associated with inflammatory lesions. Infected cells were found most consistently in the epididymis. In one animal, SIV-infected cells (presumably Langerhans cells) were present in the stratified squamous epithelium of the foreskin. Testicular atrophy and degeneration were relatively common findings. The most common lesion in the accessory glands was mild lymphocytic inflammation. In addition, atrophy of the glandular elements and interstitial fibrosis in the prostate gland were found. One animal had a granulomatous epididymitis with multinucleate giant cells that may have been a primary SIV lesion. CONCLUSIONS: The reproductive tract pathology in chronically SIV-infected male rhesus macaques and AIDS patients is similar. SIV-infected cells can be found at all levels of the male rhesus macaque reproductive tract and SIV can infect cells in the mucosal epithelium of the foreskin. This latter finding indicates that target cells for HIV transmission may be present in the foreskin epithelium of uncircumcised men.

Animals↗
PubMed1993

Characterization of T and B cells isolated from mucosa-associated tissues of the rhesus macaque.

This study has assessed T cell subsets according to the expression of CD4 and CD8 and the isotype of surface Ig+ (sIg+) B cells and plasma cells in mononuclear cells (MC) isolated from mucosa-associated tissues of rhesus macaques in comparison to lymphoid cells from systemic tissues, i.e., spleen and peripheral lymph nodes (PLN). Using enzymatic and/or mechanical dissociation methods, mononuclear cells were isolated from lamina propria (LP) of the small (jejunum and ileum) and large (cecum) intestines, parotid glands (PG), and mesenteric lymph nodes (MLN). Approximately equal numbers of CD4+ and CD8+ T cells occurred in mucosa-associated tissues (CD4:CD8 ratios of 0.9-1.2), while slightly higher numbers of CD8+ T cells were seen in spleen and PLN (CD4:CD8 ratios of 0.6-0.9). When the isotypes of sIg+ B cells were assessed in MC isolated from mucosa-associated tissues, the highest frequency of sIgA+ B cells was seen in MLN, which may represent B cells which had recently migrated from IgA inductive sites. However, IgA effector tissues, such as intestinal LP and PG, contained frequencies of sIgA+ B cells lower than those of the MLN. When Ig-producing cells were examined, 50-90% of Ig-secreting cells were of the IgA class. This suggests that sIgA+ B cells differentiate into IgA plasma cells at higher rates in the mucosal effector tissues. Plasma cells of IgM isotype were also found in significant numbers in the LP of the intestine and PG, while IgG plasma cells were most prevalent in spleen and PLN. Taken together, mucosa-associated tissues of rhesus macaques are characterized by higher numbers of CD4+ T cells and Ig-secreting plasma cells of IgA and IgM isotypes than those of systemic lymphoid tissues.

Animals↗
PubMed1993

Airflow measurements: theory and utility of findings.

The biomechanical movements of vocal structures are shown to control flow of expiratory air for the production of speech sounds. Laminar and turbulent air flow are discussed as they relate to sound generation. Theoretic and therapeutic utility of air flow profiles in differentiating linguistic contrasts and vocal characteristics are discussed along with considerations of relative utility and accuracy for various flow-measuring devices and strategies.

Adolescent↗
PubMed1993

Evidence of horizontal transmission of Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques.

This retrospective study used sensitive immunohistochemical methods to detect Pneumocystis carinii infections in fixed lung tissues collected from 107 simian immunodeficiency virus (SIV)-infected and 10 noninfected rhesus macaques during a 4-year period. P. carinii were detected in 51% of 85 terminally ill SIV-infected macaques but in only 2 of 22 macaques killed at earlier stages of SIV infection. P. carinii were not detected in any SIV-infected macaques held in isolators or in uninfected controls. Infection rates varied significantly between rooms, and the percentage of clinically important P. carinii infections increased from 0 in the first 2 years to > 50% during the final year. Infections were centered on terminal airways in 59% of infected animals. These results challenge the assumption that P. carinii pneumonia (PCP) results primarily from reactivated latent infections and suggest instead that horizontal spread of infection is important in the epidemiology of PCP in immunosuppressed macaques.

Animals↗
PubMed1993

Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.

To identify viral determinants of simian immunodeficiency virus (SIV) virulence, two pairs of reciprocal recombinants constructed from a pathogenic (SIVmac239) and a nonpathogenic (SIVmac1A11) molecular clone of SIV were tested in rhesus macaques. A large 6.2-kb fragment containing gag, pol, env, and the regulatory genes from each of the cloned (parental) viruses was exchanged to produce one pair of recombinant viruses (designated SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11gag-env/239 to indicate the genetic origins of the 5'/internal/3' regions, respectively, of the virus). A smaller 1.4-kb fragment containing the external env domain of each of the parental viruses was exchanged to create the second pair (SIVmac1A11/239env/1A11 and SIVmac239/1A11env/239) of recombinant viruses. Each of the two parental and four recombinant viruses was inoculated intravenously into four rhesus macaques, and all 24 animals were viremic by 4 weeks postinoculation (p.i.). Virus could not be isolated from peripheral blood mononuclear cells (PBMC) of any animals infected with SIVmac1A11 after 6 weeks p.i. but was consistently isolated from all macaques inoculated with SIVmac239 for 92 weeks p.i. Virus isolation was variable from animals infected with recombinant viruses; SIVmac1A11/239gag-env/1A11 and SIVmac239/1A11env/239 were isolated most frequently. Animals inoculated with SIVmac239 had 10 to 100 times more virus-infected PBMC than those infected with recombinant viruses. Three animals infected with SIVmac239 died with simian AIDS (SAIDS) during the 2-year observation period after inoculation, and the fourth SIVmac239-infected animal had clinical signs of SAIDS. Two animals infected with recombinant viruses died with SAIDS; one was infected with SIVmac239/1A11gag-env/239, and the other was infected with SIVmac1A11/239gag-env/1A11. The remaining 18 macaques remained healthy by 2 years p.i., and 13 were aviremic. One year after inoculation, peripheral lymph nodes of some of these healthy, aviremic animals harbored infected cells. All animals seroconverted within the first few weeks of infection, and the magnitude of antibody response to SIV was proportional to the levels and duration of viremia. Virus-suppressive PBMC were detected within 2 to 4 weeks p.i. in all animals but tended to decline as viremia disappeared. There was no association of levels of cell-mediated virus-suppressive activity and either virus load or disease progression. Taken together, these results indicate that differences in more than one region of the viral genome are responsible for the lack of virulence of SIVmac1A11.

Animals↗
PubMed1993

Comparison of an enzyme immuno assay (Antigenz Chlamydia) with cell culture for the detection of genital chlamydial infection in high and low risk populations.

OBJECTIVE: To evaluate a new enzyme immuno assay, Antigenz Chlamydia, for the detection of chlamydial antigen in urogenital samples using cell culture as the gold standard for comparison and direct immuno fluorescence to aid resolving the discrepancies. SUBJECTS: 212 men and 303 women attending an STD clinic and 404 women of child bearing age attending gynaecological clinics. RESULTS: Sensitivity, specificity, predictive value of positive result and predictive value of negative result of the new test were 77.6%, 99.0%, 94.3%, and 95.7% respectively in the high risk population with a prevalence of 16.5%. In the low risk population the figures were 84.2%, 94.5%, 43.2% and 99.2% respectively with a prevalence of 4.7%. CONCLUSION: Antigenz Chlamydia is an easy and quick test to perform. The test is suitable for use in high risk and low risk populations provided all positive samples are confirmed.

Adult↗
PubMed1993

The endogenous inhibitor of protein kinase-C in the rat ovary is a protein phosphatase.

Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase.

Animals↗