Prevalence of Staphylococcus aureus colonization among high-risk HIV populations

Staphylococcus aureus is a common microorganism in humans, typically colonizing the nasopharynx, skin and other mucosal surfaces. It is among the most frequent causes of clinically-significant bacterial infections accounting for increased morbidity and mortality among individuals with HIV/AIDS. Evidence of higher colonization rates among high-risk HIV populations have been observed however, prevalence estimates have varied. Additionally, behavioral, biological, and/or environmental factors that may account for these high colonization rates are not understood. Previous literature on clinic-based surveys were subject to considerable biases. Additionally, representative samples of high-risk HIV populations were difficult to obtain due in part to an underrepresentation of individuals who may not regularly obtain health care. ^ The main objective of this project is to determine the prevalence of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant (MRSA) nasal colonization in two populations: 1) men who have sex with men (MSM) and 2) injection drug users (IDU). Both of these populations are included in the third round of the National HIV Behavioral Surveillance System (NHBS) in Houston, Texas. ^ In the NHBS-MSM3 study, logistic regression was used to report odds ratios and 95% confidence intervals (CI). For the NHBS-IDU3 study, to account for the lack of independence between samples, the method of generalized estimating equations was utilized to report adjusted odds ratios and 95% CI. The NHBS-MSM3 study enrolled 202 participants with a MSSA colonization rate of 26.7% and MRSA rate of 3%. In the NHBS-IDU3 study, 18.4% were nasally colonized with MSSA and 5.7% were nasally colonized with MRSA. Among the NHBS-MSM3 population, high-risk sexual practices were associated with colonization. For the NHBS-IDU3 population, age, marital status, employment status, and the presence of scabs, were associated with colonization status when controlling for size of recruitment network. In multivariate GEE analyses, the use of antiretroviral medications and age remained significantly associated with S. aureus nasal colonization when controlling for size of recruitment network and gender. In both studies, a significantly higher than expected S. aureus and MRSA colonization rate was observed as compared to colonization rates described for the general population. However, these estimates were moderate in comparison to reported clinic-based MSM and IDU S. aureus colonization findings. This study validates substantial prevalence differences and biases that may exist with data collected from clinic-based MSM and IDU. The prevalence of MSSA and MRSA nasal colonization did not differ significantly with respect to HIV status among NHBS-MSM3/NHBS-IDU3 participants. Continued examination on the effects of S. aureus colonization and infection should be examined longitudinally to confirm additional community-based determinants in populations that are disproportionately affected.^

Optimal, minimax, and Bayesian two-stage designs in two-dose phase II clinical trials

Background: For most cytotoxic and biologic anti-cancer agents, the response rate of the drug is commonly assumed to be non-decreasing with an increasing dose. However, an increasing dose does not always result in an appreciable increase in the response rate. This may especially be true at high doses for a biologic agent. Therefore, in a phase II trial the investigators may be interested in testing the anti-tumor activity of a drug at more than one (often two) doses, instead of only at the maximum tolerated dose (MTD). This way, when the lower dose appears equally effective, this dose can be recommended for further confirmatory testing in a phase III trial under potential long-term toxicity and cost considerations. A common approach to designing such a phase II trial has been to use an independent (e.g., Simon's two-stage) design at each dose ignoring the prior knowledge about the ordering of the response probabilities at the different doses. However, failure to account for this ordering constraint in estimating the response probabilities may result in an inefficient design. In this dissertation, we developed extensions of Simon's optimal and minimax two-stage designs, including both frequentist and Bayesian methods, for two doses that assume ordered response rates between doses. ^ Methods: Optimal and minimax two-stage designs are proposed for phase II clinical trials in settings where the true response rates at two dose levels are ordered. We borrow strength between doses using isotonic regression and control the joint and/or marginal error probabilities. Bayesian two-stage designs are also proposed under a stochastic ordering constraint. ^ Results: Compared to Simon's designs, when controlling the power and type I error at the same levels, the proposed frequentist and Bayesian designs reduce the maximum and expected sample sizes. Most of the proposed designs also increase the probability of early termination when the true response rates are poor. ^ Conclusion: Proposed frequentist and Bayesian designs are superior to Simon's designs in terms of operating characteristics (expected sample size and probability of early termination, when the response rates are poor) Thus, the proposed designs lead to more cost-efficient and ethical trials, and may consequently improve and expedite the drug discovery process. The proposed designs may be extended to designs of multiple group trials and drug combination trials.^

Azithromycin efficacy in treatment of Chlamydia trachomatis

Adolescents 15 – 19 years of age have the highest prevalence of Chlamydia trachomatis out of any age group, reaching 28.3% among detained youth [1]. The 2010 Center for Disease Control guidelines recommend one dose of azithromycin for the treatment of uncomplicated chlamydia infections based on 97% cure rate with azithromycin. Recent studies found an 8% or higher failure rate of azithromycin treatment in adolescents [2-5]. We conducted a prospective study beginning May, 2012 in the Harris County Juvenile Justice Center (HCJJC) medical department. Study subjects were detainees with positive urine NAAT tests for chlamydia on intake. We provided treatment with Azithromycin, completed questionnaires assessing risk factors and performed a test of cure for chlamydia three weeks after successful treatment. Those with treatment failure (positive TOC) received doxycycline for seven days. The preliminary results summarized herein are based on data collected from May 2012 to January 2013. Of the 97 youth enrolled in the study to date, 4 (4.1%) experienced treatment failure after administration of Azithromycin. Of these four patients, all were male, African-American and asymptomatic at the time of initial diagnosis and treatment. Of note, 37 (38%) patients in the cohort complained of abdominal pain with administration of Azithromycin. Results to date suggest that the efficacy of Azithromycin in our study is higher than the recent reported studies indicating a possible upper bound of Azithromycin. These results are preliminary and recruitment will continue until a sample size of 127 youth is reached.^

Evaluation of memory B cells among perinatally HIV infected children from Houston Texas

Background: HIV associated B cell exhaustion is a notable characteristic of HIV viremic adults. However, it is not known if such alterations are present in perinatal HIV infected children, whose viral dynamics differs from those seen in adults. In the present study we perform an analysis of B cells subsets and measure antigen-specific memory B cells (MBC) in a pediatric HIV infected cohort. ^ Methods: Peripheral mononuclear cells (PBMC) of perinatal HIV infected individuals are characterized into naïve (CD21hi/CD27−), classic (CD27+), tissue like (CD21lo/CD27 −) and activated MBC (CD27+CD21− ) by FACS. A memory ELISPOT assay is used to detect antibody secreting cells. We measure total IgG and antibodies specific for influenza, HBV, mumps, measles, rubella and VZV. Memory was expressed as spot forming cells (SPC) /million of PBMC. Wilcoxon rank-sum was used to compare unpaired groups and linear regression analysis was used to determine predictors of B cell dysfunction ^ Results: 41 HIV perinatal infected children are included (51.2% females and 65.9% Black). Age at study is median (range) 8.78 years (4.39-11.57). At the time of testing they have a CD4% of 30.9 (23.2-39.4), a viral load (VL) of 1.95 log10 copies/ml (1.68-3.29) and a cumulative VL of 3.4 log10 copy × days (2.7-4.0). Ninety two percent of the children are on cARV for > 6 months. Overall, HIV+ children compared with controls have a significant lower number of IgG and antigen specific SFC. In addition, they have a lower proportion of classical MBC 12.9 (8.09-19.85) vs 29.4 (18.7-39.05); 0.01, but a significant higher proportion of tissue like memory MBC 6.01 (2.79-12.7) vs 0.99 (0.87-1.38); 0.003, compared with controls. Patients are parsed on VL (<400 and ≥ 400 copies/ml) with the objective to evaluate the effect of VL on B cell status. Patients with a VL ≥ 400 copies/ml have a significantly lower IgG, HBV, measles, rubella and VZV SPC compared with those with a VL < 400 copies/ml. There are no significant differences in B cell subpopulations between the groups. A moderate negative correlation was observed between the time of cARV initiation and the frequency of IgG memory B cells, suggesting that early initiation of cARV appears to lead to a better functionality of the IgG memory B cells (P=0.05). A statistically significant positive correlation was observed between the total number of IgG memory cells and the number of antigen-specific memory B cells/SPCs. Suggesting that the progressive recovery of the IgG memory B cell pull goes along with a progressive increase in the number of antigen-specific SPCs. ^ Conclusion: A pediatric cohort in overall good status with respect to HIV infection and on ART has defects in B cell function and numbers (reduced total and antigen specific MBC and increased tissue like and reduced classical MBC).^

Study on the effect of metformin in patients of metastatic renal cell cancer with diabetes receiving tyrosine kinase inhibitor therapy

Objective: The primary objective of our study was to study the effect of metformin in patients of metastatic renal cell cancer (mRCC) and diabetes who are on treatment with frontline therapy of tyrosine kinase inhibitors. The effect of therapy was described in terms of overall survival and progression free survival. Comparisons were made between group of patients receiving metformin versus group of patients receiving insulin in diabetic patients of metastatic renal cancer on frontline therapy. Exploratory analyses were also done comparing non-diabetic patients of metastatic renal cell cancer receiving frontline therapy compared to diabetic patients of metastatic renal cell cancer receiving metformin therapy. ^ Methods: The study design is a retrospective case series to elaborate the response rate of frontline therapy in combination with metformin for mRCC patients with type 2 diabetes mellitus. The cohort was selected from a database, which was generated for assessing the effect of tyrosine kinase inhibitor therapy associated hypertension in metastatic renal cell cancer at MD Anderson Cancer Center. Patients who had been started on frontline therapy for metastatic renal cell carcinoma from all ethnic and racial backgrounds were selected for the study. The exclusion criteria would be of patients who took frontline therapy for less than 3 months or were lost to follow-up. Our exposure variable was treatment with metformin, which comprised of patients who took metformin for the treatment of type 2 diabetes at any time of diagnosis of metastatic renal cell carcinoma. The outcomes assessed were last available follow-up or date of death for the overall survival and date of progression of disease from their radiological reports for time to progression. The response rates were compared by covariates that are known to be strongly associated with renal cell cancer. ^ Results: For our primary analyses between the insulin and metformin group, there were 82 patients, out of which 50 took insulin therapy and 32 took metformin therapy for type 2 diabetes. For our exploratory analysis, we compared 32 diabetic patients on metformin to 146 non-diabetic patients, not on metformin. Baseline characteristics were compared among the population. The time from the start of treatment until the date of progression of renal cell cancer and date of death or last follow-up were estimated for survival analysis. ^ In our primary analyses, there was a significant difference in the time to progression of patients receiving metformin therapy vs insulin therapy, which was also seen in our exploratory analyses. The median time to progression in primary analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 540 days (95% CI: 350-894) in patients who were receiving insulin therapy (p=0.024). The median time to progression in exploratory analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 279 days (95% CI: 202-372 days) in non-diabetic group (p-value <0.0001). ^ The median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 816 days (95%CI: 558-1405 days) in insulin group (p-value<0.91). For the exploratory analyses, the median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 766 days (95%CI: 649-965 days) in the non-diabetic group (p-value<0.78). Metformin was observed to increase the progression free survival in both the primary and exploratory analyses (HR=0.52 in metformin Vs insulin group and HR=0.36 in metformin Vs non-diabetic group, respectively). ^ Conclusion: In laboratory studies and a few clinical studies metformin has been proven to have dual benefits in patients suffering from cancer and type 2-diabetes via its action on the mammalian target of Rapamycin pathway and effect in decreasing blood sugar by increasing the sensitivity of the insulin receptors to insulin. Several studies in breast cancer patients have documented a beneficial effect (quantified by pathological remission of cancer) of metformin use in patients taking treatment for breast cancer therapy. Combination of metformin therapy in patients taking frontline therapy for renal cell cancer may provide a significant benefit in prolonging the overall survival in patients with metastatic renal cell cancer and diabetes. ^

Indeterminate QunatiFERON-TB Gold In-Tube assay results in association with procedural specimen collection in children

BACKGROUND. The development of interferon-gamma release assays (IGRA) has introduced powerful tools in diagnosing latent tuberculosis infection (LTBI) and may play a critical role in the future of tuberculosis diagnosis. However, there have been reports of high indeterminate results in young patient populations (0-18 years). This study investigated results of the QunatiFERON-TB Gold In-Tube (QFT-GIT) IGRA in a population of children (0-18 years) at Texas Children's Hospital in association with specimen collection procedures using surrogate variables. ^ METHODS. A retrospective case-control study design was used for this investigation. Cases were defined as having QFT-GIT indeterminate results. Controls were defined as having either positive or negative results (determinates). Patients' admission status, staff performing specimen collection, and specific nurse performing specimen collection were used as surrogates to measure specimen collection procedures. ^ To minimize potential confounding, abstraction of patients' electronic medical records was performed. Abstracted data included patients' medications and evaluation at the time of QFT-GIT specimen collection in addition to their medical history. QFT-GIT related data was also abstracted. Cases and controls were characterized using chi-squared tests or Fisher's exact tests across categorical variables. Continuous variables were analyzed using one-way ANOVA and t-tests for continuous variables. A multivariate model was constructed by backward stepwise removal of statistically significant variables from univariate analysis. ^ RESULTS. Patient data was abstracted from 182 individuals aged 0-18 years from July 2010 to August 2011 at Texas Children's Hospital. 56 cases (indeterminates) and 126 controls (determinates) were enrolled. Cancer was found to be an effect modifier with subsequent stratification resulting in a cancer patient population too small to analyze (n=13). Subsequent analyses excluded these patients. ^ The exclusion of cancer patients resulted in a population of 169 patients with 49 indeterminates (28.99%) and 120 determinates (71.01%), with mean ages of 9.73 (95% CI: 8.03, 11.43) years and 11.66 (95% CI: 10.75, 12.56) years (p = 0.033), respectively. Median age of patients who were indeterminates and determinates were 12.37 and 12.87 years, respectively. Lack of data for our specific nurse surrogate (QFTNurse) resulted in its exclusion from analysis. The final model included only our remaining surrogate variables (QFTStaff and QFTInpatientOutpatient). The staff collecting surrogate (QFTStaff) was found to be modestly associated with indeterminates when nurses collected the specimen (OR = 1.54, 95% CI: 0.51, 4.64, p = 0.439) in the final model. Inpatients were found to have a strong and statistically significant association with indeterminates (OR = 11.65, 95% CI: 3.89, 34.9, p < 0.001) in the final model. ^ CONCLUSION. Inpatient status was used as a surrogate for indication of nurse drawn blood specimens. Nurses have had little to no training regarding shaking of tubes versus phlebotomists regarding QFT-GIT testing procedures. This was also measured by two other surrogates; specifically a medical note stating whether a nurse or phlebotomist collected the specimen (QFTStaff) and the name and title of the specific nurse if collection was performed by a nurse (QFTNurse). Results indicated that inpatient status was a strong and statistically significant factor for indeterminates, however, nurse collected specimens and indeterminate results had no statistically significant association in non-cancer patients. The lack of data denoting the specific nurse performing specimen collection excluded the QFTNurse surrogate in our analysis. ^ Findings suggests training of staff personnel in specimen procedures may have little effect on the number of indeterminates while inpatient status and thus possibly illness severity may be the most important factor for indeterminate results in this population. The lack of congruence between our surrogate measures may imply that our inpatient surrogate gauged illness severity rather than collection procedures as intended. ^ Despite the lack of clear findings, our analysis indicated that more than half of indeterminates were found in specimens drawn by nurses and as such staff training may be explored. Future studies may explore methods in measuring modifiable variables during pre-analytical QFT-GIT procedures that can be discerned and controlled. Identification of such measures may provide insight into ways to lowering indeterminate QFT-GIT rates in children.^

Assessing the patterns of colorectal cancer treatment, adherence to treatment guidelines and survival in Texas

Colorectal cancer (CRC) is the third leading cancer in both incidence and mortality in Texas. This study investigated the adherence of CRC treatment to standard treatment guidelines and the association between standard treatment and CRC survival in Texas. The author used Texas Cancer Registry (TCR) and Medicare linked data to study the CRC treatment patterns and factors associated with standard treatment in patients who were more than 65 years old and were diagnosed in 2001 through 2007. We also determined whether adherence to standard treatment affect patients' survival. Multiple logistic regression and Cox regression analysis were used to analyze our data. Both regression models are adjusted for demographic characteristics and tumor characteristics. We found that for the 3977 regional colon cancer patients 80 years old or younger, 60.2% of them received chemotherapy, in adherence to the recommended treatment guidelines. People with younger age, female gender, higher education and lower comorbidity score are more likely adherent to this surgery guideline. Patients' adherence to chemotherapy in this cohort have better survival compared to those who are not (HR: 0.76, 95% CI: 0.68-0.84). For the 12709 colon cancer patients treated with surgery, 49.3% have more than 12 lymph nodes removed, in adherence to the treatment guidelines. People with younger age, female gender, higher education, regional stage, lager tumor size and lower comorbidity score are more likely to adherent to this surgery guideline. Patients with more than 12 lymph nodes removed in this cohort have better survival (HR: 0.86, 95% CI: 0.82-0.91). For the 1211 regional rectal cancer patients 80 years old or younger, 63.2% of them were adherent to radiation treatment. People with smaller tumor size and lower comorbidity score are more likely to adherent to this radiation guideline. There is no significant survival difference between radiation adherent patients and non-adherent patients (HR: 1.03, 95% CI: 0.82-1.29). For the 1122 regional rectal cancer patients 80 years old or younger who were treated with surgery, 76.0% of them received postoperative chemotherapy, in adherence to the treatment guidelines. People with younger age and smaller comorbidity score are related with higher adherence rate. Patients adherent with adjuvant chemotherapy in this cohort have better survival than those were not adherent (HR: 0.60, 95% CI: 0.45-0.79).^

Weight-for-age percentile and its association to community acquired methicillin resistant Staphylococcus aureus among pediatric patients in Houston, TX

BACKGROUND: Weight has been implicated as a risk factor for symptomatic community-acquired methicillin resistant Staphylococcus Aureus (CA-MRSA). Information from Texas Children's Hospital (TCH) in Houston, TX was used to implement a case-control study to assess weight-for-age percentile (WFA), race and seasonal exposure as risk factors. ^ METHODS: A retrospective chart review to collect data from TCH was conducted covering the time period January 1st, 2008 to May 31st, 2011. Cases were confirmed and identified by the infectious disease department and were matched on a 1:1 ratio to controls that were seen by the emergency department for non-infected fractures from June 1st, 2008 to May 31st, 2011. Data abstraction was performed using TCH's electronic medical records (EMR) system (EPIC ®). ^ RESULTS: Of 702 CA-MRSA identified cases, ages 9 to 16.99, 564 (80.3%) had the variable `weight' present in their EMR, were not duplicates and not determined to be outliers. Cases were randomly matched to a pool of available controls (n=1864) according to age and gender, yielding 539 1:1 matched pairs (95.5% case matching success) with a total study sample size, N=1078. Case median age was 13.38 years with the majority being White (66.05%) and male (59.4%). Adjusted conditional logistic regression analysis of the matched pairs identified the following risk factors to presenting with CA-MRSA infection among pediatric patients, ages 9 to 16.99 years: a) Individual weight in the highest (75th-99.9th) WFA quartile (OR=1.36; 95% confidence interval [CI]=1.06-1.74; P= 0.016), b) Infection during summer months (OR: 1.69; 95% CI=1.2-2.38; P= 0.003), c) patients of African American race/ethnicity (OR= 1.48; 95% CI=1.13-1.95; P= 0.004). ^ CONCLUSIONS: Pediatric patients, 9 to 16.99 years of age, in the highest WFA quartile (75th-99.9th), or of African-American race had an associated increased risk of presenting with CA-MRSA infection. Furthermore, children in this population were at a higher risk of contracting CA-MRSA infection during the summer season.^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^

Age -period -cohort analysis: An extension of Cox's lifetable regression with time -dependent covariates

It is well known that an identification problem exists in the analysis of age-period-cohort data because of the relationship among the three factors (date of birth + age at death = date of death). There are numerous suggestions about how to analyze the data. No one solution has been satisfactory. The purpose of this study is to provide another analytic method by extending the Cox's lifetable regression model with time-dependent covariates. The new approach contains the following features: (1) It is based on the conditional maximum likelihood procedure using a proportional hazard function described by Cox (1972), treating the age factor as the underlying hazard to estimate the parameters for the cohort and period factors. (2) The model is flexible so that both the cohort and period factors can be treated as dummy or continuous variables, and the parameter estimations can be obtained for numerous combinations of variables as in a regression analysis. (3) The model is applicable even when the time period is unequally spaced.^ Two specific models are considered to illustrate the new approach and applied to the U.S. prostate cancer data. We find that there are significant differences between all cohorts and there is a significant period effect for both whites and nonwhites. The underlying hazard increases exponentially with age indicating that old people have much higher risk than young people. A log transformation of relative risk shows that the prostate cancer risk declined in recent cohorts for both models. However, prostate cancer risk declined 5 cohorts (25 years) earlier for whites than for nonwhites under the period factor model (0 0 0 1 1 1 1). These latter results are similar to the previous study by Holford (1983).^ The new approach offers a general method to analyze the age-period-cohort data without using any arbitrary constraint in the model. ^

Calmodulin -regulated processes and intracellular calcium in the heat stress response of mammalian cells

Three approaches were used to examine the role of Ca$\sp{2+}$- and/or calmodulin (CaM)-regulated processes in the mammalian heat stress response. The focus of the first approach was on the major Ca$\sp{2+}$-binding protein, CaM, and involved the use of CaM antagonists that perturbed CaM-regulated processes during heat stress. The second approach involved the use of a cell line and its BPV-1 transformants that express increased basal levels of CaM, or parvalbumin--a Ca$\sp{2+}$-binding protein not normally found in these cells. The last approach used Ca$\sp{2+}$ chelators to buffer Ca$\sp{2+}$-transients.^ The principle conclusions resulting from these three experimental approaches are: (1) CaM antagonists cause a temperature-dependent potentiation of heat killing, but do not inhibit the triggering and development of thermotolerance suggesting some targets for heat killing are different from those that lead to thermotolerance; (2) Members of major HSP families (especially HSP70) can bind to CaM in a Ca$\sp{2+}$-dependent manner in vitro, and HSP have been associated with events leading to thermotolerance. But, because thermotolerance is not affected by CaM antagonists, and antagonists should interfere with HSP binding to CaM, the events leading to triggering or developing thermotolerance were not strongly dependent on HSP binding to CaM; (3) CaM antagonists can also bind to HSP70 (and possibly other HSP) suggesting an alternative mechanism for the action of these agents in heat killing may involve direct binding to other proteins, like HSP70, whose function is important for survival following heating and inhibiting their activity; and (4) The signal governing the rate of synthesis of another major HSP group, the HSP26 family, can be largely abrogated by elevated Ca$\sp{2+}$-binding proteins or Ca$\sp{2+}$ chelators without significantly reducing survival or thermotolerance suggesting if the HSP26 family is involved in either end point, it may function in (Ca$\sp{2+}$) $\sb{\rm i}$ homeostasis. ^

Estrogen induced desensitization of c -{\it fos\/} messenger RNA expression {\it in vivo\/}

In this thesis, we investigated the regulation of the nuclear proto-oncogene, c-fos by estrogen in vivo. In the uterus, estrogen causes a rapid, dramatic and transient induction of c-fos mRNA and this occurs by transcriptional activation. We have discovered a previously unrecognized regulatory mechanism by which fos becomes desensitized to estrogen following the transient induction. We investigated three aspects of this desensitization: (1) the kinetics and general characteristics of the phenomenon; (2) the molecular mechanism of the desensitization; and (3) the relationship of desensitization to estrogen stimulated DNA synthesis. The desensitization occurs between 3-24 hours after initial hormonal stimulation and is reversible within 72 hours. The desensitization is not species specific, in that it occurs in both the rat and mouse. The desensitization also occurs in at least two estrogen responsive tissues, the uterus and vagina. The desensitization is not unique to c-fos, since both c-myc and c-jun show similar patterns of desensitization. However, the desensitization is not observed with creatine kinase B (CKB), indicating that not all estrogen inducible genes become desensitized. In the second general area, we determined the desensitization is at the transcriptional level. The desensitization is homologous, but not heterologous, since estrogen induction does not desensitize c-fos to other agents. Other studies show that the desensitization is not due to the lack of functional estrogen receptors. Taken together, these findings suggest that the desensitization occurs at the level of the estrogen responsive element. In the third major area, we demonstrated that the desensitization appears to be related to estrogen induced DNA synthesis. Support for this suggestion comes from the observation that short acting estrogens which induce fos, but not DNA synthesis, do not produce desensitization. ^

Regulation of {\it neu\/} gene expression by the simian virus 40 large T antigen and tumor suppressors Rb and p53

The neu gene (also c-erbB-2 or HER2) encodes a 185 kilodalton protein that is frequently overexpressed in breast, ovarian and non-small cell lung cancers. Study of the regulation of neu indicates that neu gene expression can be modulated by c-myc or by the adenovirus 5 E1a gene product. This study demonstrates that the transforming protein, large T antigen, of the simian virus 40 represses neu promoter activity. Repression of neu by large T antigen is mediated through the region $-$172 to $-$79 (relative to first ATG) of the neu promoter--unlike through $-$312 to $-$172 for c-myc or E1a. This suggests a different pathway for repression of neu by large T antigen. The 10 amino acid region of large T required for binding the tumor suppressor, retinoblastoma gene product, Rb, is not necessary for repression of neu. Moreover, the tumor suppressors, Rb and p53 can independently inhibit neu promoter activity. Rb inhibits neu through a 10 base pair G-rich enhancer (GTG element) ($-$243 to $-$234) and also through regions close to transcription initiation sites ($-$172 to $-$79). Mutant Rb unable to complex large T is able to repress the region close to transcription initiation but not the GTG enhancer. Thus, Rb inhibits the two regulatory domains of the neu gene by different mechanisms. Both Rb and p53 can repress the transforming activity of activated neu in focus forming assays. These data provide evidence that tumor suppressors regulate expression of growth stimulatory genes such as neu. Therefore, one reason for the overexpression of neu that is frequently seen in breast cancer cells may be due to functional inactivation of Rb and p53 which is also a common occurrence in breast cancer cells. ^

Molecular studies of human high grade B cell non -Hodgkin's lymphomas

The non-Hodgkin's B cell lymphomas are a diverse group of neoplastic diseases. The incidence rate of the malignant tumors has been rising rapidly over the past twenty years in the United States and worldwide. The lack of insight to pathogenesis of the disease poses a significant problem in the early detection and effective treatment of the human malignancies. These studies attempted to investigate the molecular basis of pathogenesis of the human high grade B cell non-Hodgkin's lymphomas with a reverse genetic approach. The specific objective was to clone gene(s) which may play roles in development and progression of human high grade B cell non-Hodgkin's lymphomas.^ The messenger RNAs from two high grade B cell lymphoma lines, CJ and RR, were used for construction of cDNA libraries. Differential screening of the derived cDNA libraries yielded a 1.4 kb cDNA clone. The gene, designated as NHL-B1.4, was shown to be highly amplified and over-expressed in the high grade B cell lymphoma lines. It was not expressed in the peripheral blood lymphoid cells from normal donors. However, it was inducible in peripheral blood T lymphocytes by a T cell mitogen, PHA, but could not be activated in normal B cells by B cell mitogen PMA. Further molecular characterization revealed that the gene may have been rearranged in the RR and some other B cell lymphoma lines. The coding capacity of the cDNA has been confirmed by a rabbit reticulocyte lysate and wheat germ protein synthesis system. A recombinant protein with a molecular weight of approximate 30 kDa was visualized in autoradiogram. Polyclonal antisera have been generated by immunization of two rabbits with the NHL-B1.4 recombinant protein produced in the E. coli JM109. The derived antibody can recognize a natural protein with molecular weight of 49 kDa in cell lysate of activated peripheral T lymphocytes of normal donors and both the cell lysate and supernatant of RR B cell lymphoma lines. The possible biologic functions of the molecule has been tested preliminarily in a B lymphocyte proliferation assay. It was found that the Q-sepharose chromatograph purified supernatant of COS cell transfection could increase tritiated thymidine uptake by B lymphocytes but not by T lymphocytes. The B cell stimulatory activity of the supernatant of COS cell tranfection could be neutralized by the polyclonal antisera, indicating that the NHL-B1.4 gene product may be a molecule with BCGF-like activity.^ The expression profiles of NHL-B1.4 in normal and neoplastic lymphoid cells were consistent with the current B lymphocyte activation model and autocrine hypothesis of high grade B cell lymphomagenesis. These results suggested that the NHL-B1.4 cDNA may be a disease-related gene of human high grade B cell lymphomas, which may codes for a postulated B cell autocrine growth factor. ^

Expression of p53, {\it * ras\/} and PCNA in human colonic neoplasms: Possible biomarkers of malignant potential

Colorectal cancer is a leading cause of cancer mortality and early detection can significantly improve the clinical outcome. Most colorectal cancers arise from benign neoplastic lesions recognized as adenomas. Only a small percentage of all adenomas will become malignant. Thus, there is a need to identify specific markers of malignant potential. Studies at the molecular level have demonstrated an accumulation of genetic alterations, some hereditary but for the most occurring in somatic cells. The most common are the activation of ras, an oncogene involved in signal transduction, and the inactivation of p53, a tumor suppressor gene implicated in cell cycle regulation. In this study, 38 carcinomas, 95 adenomas and 20 benign polyps were analyzed by immunohistochemistry for the abnormal expression of p53 and ras proteins. An index of cellular proliferation was also measured by labeling with PCNA. A general overexpression of p53 was immunodetected in 66% of the carcinomas, while 26% of adenomas displayed scattered individual positive cells or a focal high concentration of positive cells. This later was more associated with severe dysplasia. Ras protein was detected in 37% of carcinomas and 32% of adenomas mostly throughout the tissue. p53 immunodetection was more frequent in adenomas originating in colons with synchronous carcinomas, particularly in patients with familial adenomatous polyposis and it may be a useful marker in these cases. Difference in the frequency of p53 and ras alterationbs was related to the location of the neoplasm. Immunodetection of p53 protein was correlated to the presence of a mutation in p53 gene at exon 7 and 5 in 4/6 carcinomas studied and 2 villous adenomas. Thus, we characterized in adenomas the abnormal expression of two proteins encoded by the most commonly altered genes in colorectal cancer. p53 alteration appears to be more specifically associated with transition to malignancy than ras. By using immunohistochemistry, a technique that keeps the architecture of the tissue intact, it was possible to correlate these alterations to histopathological characteristics that were associated with higher risks for transformation: villous content, dysplasia and size of adenoma. ^