Deep Brain Stimulation of Bilateral Centromedian Thalamic Nuclei in Pediatric Patients with Lennox-Gastaut Syndrome: An Institutional Experience.

BACKGROUND: Surgical management of pediatric patients with nonlesional, drug-resistant epilepsy, including patients with Lennox-Gastaut syndrome (LGS), remains a challenge given the lack of resective targets in most patients and shows seizure freedom rates <50% at 5 years. The efficacy of deep brain stimulation (DBS) is less certain in children than in adults. This study examined clinical and seizure outcomes for pediatric patients with LGS undergoing DBS targeting of the centromedian thalamic nuclei (CMTN). METHODS: An institutional review board-approved retrospective analysis was performed of patients aged ≤19 years with clinical diagnosis of LGS undergoing bilateral DBS placement to the CMTN from 2020 to 2021 by a single surgeon. RESULTS: Four females and 2 males aged 6-19 years were identified. Before surgery, each child experienced at least 6 years of refractory seizures; 4 children had experienced seizures since infancy. All took antiseizure medications at the time of surgery. Five children had previous placement of a vagus nerve stimulator and 2 had a previous corpus callosotomy. The mean length of stay after DBS was 2 days. No children experienced adverse neurologic effects from implantation; the mean follow-up time was 16.3 months. Four patients had >60% reduction in seizure frequency after surgery, 1 patient experienced 10% reduction, and 1 patient showed no change. No children reported worsening seizure symptoms after surgery. CONCLUSIONS: Our study contributes to the sparse literature describing CMTN DBS for children with drug-resistant epilepsy from LGS. Our results suggest that CMTN DBS is a safe and effective therapeutic modality that should be considered as an alternative or adjuvant therapy for this challenging patient population. Further studies with larger patient populations are warranted.

Liposomal Glutathione Supplementation Mitigates Extrapulmonary Tuberculosis in the Liver and Spleen.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) accounts for a fifth of all Mycobacterium tuberculosis (M. tb) infections worldwide. The rise of multidrug resistance in M. tb alongside the hepatotoxicity associated with antibiotics presents challenges in managing and treating tuberculosis (TB), thereby prompting a need for new therapeutic approaches. Administration of liposomal glutathione (L-GSH) has previously been shown to lower oxidative stress, enhance a granulomatous response, and reduce the burden of M. tb in the lungs of M. tb-infected mice. However, the effects of L-GSH supplementation during active EPTB in the liver and spleen have yet to be explored. METHODS: In this study, we evaluated hepatic glutathione (GSH) and malondialdehyde (MDA) levels, and the cytokine profiles of untreated and L-GSH-treated M. tb-infected wild type (WT) mice. Additionally, the hepatic and splenic M. tb burdens and tissue pathologies were also assessed. RESULTS: L-GSH supplementation increased total hepatic levels and reduced GSH. A decrease in the levels of MDA, oxidized GSH, and interleukin (IL)-6 was also detected following L-GSH treatment. Furthermore, L-GSH supplementation was observed to increase interferon-gamma (IFN-γ) and tumor necrosis factor (TNF)-α production and decrease IL-10 levels. M. tb survival was significantly reduced in the liver and spleen following L-GSH supplementation. L-GSH treatment also provided a host-protective effect in the liver and spleen of M. tb-infected mice. CONCLUSIONS: Overall, L-GSH supplementation elevated the levels of total and reduced forms of GSH in the liver and reduced the burden of M. tb by decreasing oxidative stress, enhancing the production of immunosupportive cytokines, and reducing the levels of immunosuppressive cytokines. These observed benefits highlight the potential of L-GSH supplementation during active EPTB and provide insight into novel therapeutic interventions against M. tb infections.

Evaluation of a Social Media Campaign Designed to Increase Awareness of Thirdhand Smoke among California Adults.

Despite a growing body of research outlining the harms of thirdhand smoke (THS), the public remains generally unaware of risks and exposure routes. This project built on past tobacco prevention campaigns and the tenants of McGuire's input-output model to implement and evaluate a seven-month Facebook-disseminated campaign seeking to improve THS awareness among California adults (n = 1087). Multilinear regression showed that THS-related knowledge (χ2[6] = 19.31, p < .01), attitude (χ2[6] = 13.88, p < .05), and efficacy (χ2[6] = 13.81, p < .05) significantly increased by the campaign's end, with messages highlighting children's health (r = .110, p < .05), pets (r = .145, p < .01), and dust reservoirs (r = .144, p < .01) as the most persuasive. Path analysis modeling found campaign recall to be associated with changes in knowledge (ß = .161, p < .01), which predicated attitude change (ß = .614, p < .001) and, in turn, behavior change (ß = .149, p < .05). Findings suggest social media campaigns should continue to educate diverse populations about new tobacco risks and that tobacco control advocates should consider integrating educational THS messages.

L-GSH Supplementation in Conjunction With Rifampicin Augments the Treatment Response to Mycobacterium tuberculosis in a Diabetic Mouse Model.

Both active tuberculosis (TB) and asymptomatic latent Mycobacterium tuberculosis (M. tb) infection (LTBI) cause significant health burdens to humans worldwide. Individuals with immunocompromising health conditions, such as Type 2 Diabetes Mellitus (T2DM), have a weakened ability to control M. tb infection and are more susceptible to reactivation of LTBI to active diseases. T2DM cases are known to have glutathione (GSH) deficiency and impaired immune cell function, including the granulomatous response to M. tb infection. We have previously reported that liposomal glutathione (L-GSH) supplementation can restore the immune cell effector responses of T2DM cases. However, the effects of L-GSH supplementation on the bactericidal activities of first-line anti-TB drug rifampicin (RIF) against M. tb infection have yet to be explored. The aim of this study is to elucidate the effects of L-GSH supplementation in conjunction with RIF treatment during an active M. tb infection in a diabetic mouse model. In this study, we evaluated total and reduced levels of GSH, cytokine profiles, malondialdehyde (MDA) levels, M. tb burden, and granulomatous response in the lungs. We show that L-GSH supplementation caused a significant reduction in M. tb burden in the lungs, decreased oxidative stress, and increased the production of IFN-γ, TNF-α, IL-17, IL-10, and TGF-ß1compared to the untreated mice. In addition, L-GSH supplementation in conjunction with RIF treatment achieved better control of M. tb infection in the lungs and significantly reduced the levels of oxidative stress compared to treatment with RIF alone. Moreover, L-GSH in conjunction with RIF significantly increased TGF-ß1 levels compared to treatment with RIF alone. These findings suggest potential therapeutic benefits of L-GSH supplementation in conjunction with first-line antibiotic therapy against M. tb infection in individuals with T2DM.

Liposomal Glutathione Helps to Mitigate Mycobacterium tuberculosis Infection in the Lungs.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is responsible for causing significant morbidity and mortality, especially among individuals with compromised immune systems. We have previously shown that the supplementation of liposomal glutathione (L-GSH) reduces M. tb viability and enhances a Th-1 cytokine response, promoting granuloma formation in human peripheral blood mononuclear cells in vitro. However, the effects of L-GSH supplementation in modulating the immune responses in the lungs during an active M. tb infection have yet to be explored. In this article, we report the effects of L-GSH supplementation during an active M. tb infection in a mouse model of pulmonary infection. We determine the total GSH levels, malondialdehyde (MDA) levels, cytokine profiles, granuloma formation, and M. tb burden in untreated and L-GSH-treated mice over time. In 40 mM L-GSH-supplemented mice, an increase in the total GSH levels was observed in the lungs. When compared to untreated mice, the treatment of M. tb-infected mice with 40 mM and 80 mM L-GSH resulted in a reduction in MDA levels in the lungs. L-GSH treatment also resulted in a significant increase in the levels of IL-12, IFN-γ, IL-2, IL-17, and TNF-α in the lungs, while down-regulating the production of IL-6, IL-10, and TGF-ß in the lungs. A reduction in M. tb survival along with a decrease in granuloma size in the lungs of M. tb-infected mice was observed after L-GSH treatment. Our results show that the supplementation of mice with L-GSH led to increased levels of total GSH, which is associated with reduced oxidative stress, increased levels of granuloma-promoting cytokines, and decreased M. tb burden in the lung. These results illustrate how GSH can help mitigate M. tb infection and provide an insight into future therapeutic interventions.

The utility of cerebrospinal fluid-derived cell-free DNA in molecular diagnostics for the PIK3CA-related megalencephaly-capillary malformation (MCAP) syndrome: a case report.

The megalencephaly-capillary malformation (MCAP) syndrome is an overgrowth disorder caused by mosaic gain-of-function variants in PIK3CA It is characterized by megalencephaly or hemimegalencephaly, vascular malformations, somatic overgrowth, among other features. Epilepsy is commonly associated with MCAP, and a subset of individuals have cortical malformations requiring resective epilepsy surgery. Like other mosaic disorders, establishing a molecular diagnosis is largely achieved by screening lesional tissues (such as brain or skin), with a low diagnostic yield from peripheral tissues (such as blood). Therefore, in individuals with MCAP in whom lesional tissues are scarce or unavailable or those ineligible for epilepsy surgery, establishing a molecular diagnosis can be challenging. Here we report on the utility of cerebrospinal fluid (CSF)-derived cfDNA for the molecular diagnosis of an individual with MCAP syndrome harboring a mosaic PIK3CA variant (c.3139C > T, p.His1047Tyr). The proband presented with asymmetric megalencephaly without significant dysgyria. He did not have refractory epilepsy and was therefore not a candidate for epilepsy surgery. However, he developed diffuse large B-cell lymphoma (DLBCL) in late childhood, with four CSF samples obtained via lumbar puncture for cancer staging during which one sample was collected for cfDNA extraction and sequencing. PIK3CA variant allele fractions in CSF cell-free DNA (cfDNA), skin fibroblasts, and peripheral blood were 3.08%, 37.31%, and 2.04%, respectively. This report illustrates the utility of CSF-derived cfDNA in MCAP syndrome. Minimally invasive-based molecular diagnostic approaches utilizing cfDNA not only facilitate accurate genetic diagnosis but also have important therapeutic implications for individuals with refractory epilepsy as repurposed PI3K-AKT-MTOR pathway-inhibitors become more widely available.

Effects of Oral Liposomal Glutathione in Altering the Immune Responses Against Mycobacterium tuberculosis and the Mycobacterium bovis BCG Strain in Individuals With Type 2 Diabetes.

The World Health Organization (WHO) has identified type 2 diabetes (T2DM) as a neglected, important, and re-emerging risk factor for tuberculosis (TB), especially in low and middle-income countries where TB is endemic. In this clinical trial study, oral liposomal glutathione supplementation (L-GSH) or placebo was given to individuals with T2DM to investigate the therapeutic effects of L-GSH supplementation. We report that L-GSH supplementation for 3 months in people with T2DM was able to reduce the levels of oxidative stress in all blood components and prevent depletion of glutathione (GSH) in this population known to be GSH deficient. Additionally, L-GSH supplementation significantly reduced the burden of intracellular mycobacteria within in vitro granulomas generated from peripheral blood mononuclear cells (PBMCs) of T2DM subjects. L-GSH supplementation also increased the levels of Th1-associated cytokines, IFN-γ, TNF-α, and IL-2 and decreased levels of IL-6 and IL-10. In conclusion our studies indicate that oral L-GSH supplementation in individuals with T2DM for three months was able to maintain the levels of GSH, reduce oxidative stress, and diminish mycobacterial burden within in vitro generated granulomas of diabetics. L-GSH supplementation for 3 months in diabetics was also able to modulate the levels of various cytokines.

Everolimus-induced effector mechanism in macrophages and survivability of Erdman, CDC1551 and HN878 strains of Mycobacterium tuberculosis infection.

With a disease as widespread and destructive as tuberculosis, more effective drugs and healthcare strategies, in addition to the current antibiotics regimen, are crucial for the enhanced well-being of millions of people suffering from the disease. Host-directed therapy is a new and emerging concept in treating chronic infectious diseases, such as tuberculosis. Repurposing of anti-cancer drugs, such as everolimus, may be an effective way to supplement the standard antibiotic treatment. Individuals with type 2 diabetes are increasingly susceptible to co-morbidities and co-infections including Mycobacterium tuberculosis, the causative agent of tuberculosis. We demonstrated in this study that in vitro everolimus treatment of granulomas from individuals with type 2 diabetes caused significant reduction in the viability of Mycobacterium tuberculosis.Further investigations revealed the effects of everolimus in targeting foamy macrophages, a macrophage phenotype that forms around granulomas, and is characterized by a higher lipid accumulation inside the cells. These foamy macrophages are thought to harbor dormant bacilli, which are potential sources of disease reactivation. Therefore, blocking foamy macrophage formation would help better killing of intracellular bacteria. Here, we report the potential of everolimus treatment to downregulate lipid content within the foamy macrophages of in vitro granulomas, thus leading to a potential decrease in the number of foamy macrophages and a more robust response to Mycobacterium tuberculosis.

The Effects of Oral Liposomal Glutathione and In Vitro Everolimus in Altering the Immune Responses against Mycobacterium bovis BCG Strain in Individuals with Type 2 Diabetes.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains a devastating infectious disease in the world. There has been a daunting increase in the incidence of Type 2 Diabetes Mellitus (T2DM) worldwide. T2DM patients are three times more vulnerable to M. tb infection compared to healthy individuals. TB-T2DM coincidence is a challenge for global health control. Despite some progress in the research, M. tb still has unexplored characteristics in successfully evading host defenses. The lengthy duration of treatment, the emergence of multi-drug-resistant strains and extensive-drug-resistant strains of M. tb have made TB treatment very challenging. Previously, we have tested the antimycobacterial effects of everolimus within in vitro granulomas generated from immune cells derived from peripheral blood of healthy subjects. However, the effectiveness of everolimus treatment against mycobacterial infection in individuals with T2DM is unknown. Furthermore, the effectiveness of the combination of in vivo glutathione (GSH) supplementation in individuals with T2DM along with in vitro treatment of isolated immune cells with everolimus against mycobacterial infection has never been tested. Therefore, we postulated that liposomal glutathione (L-GSH) and everolimus would offer great hope for developing adjunctive therapy for mycobacterial infection. L-GSH or placebo was administered to T2DM individuals orally for three months. Study subjects' blood was drawn pre- and post-L-GSH/or placebo supplementation, where Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood to conduct in vitro studies with everolimus. We found that in vitro treatment with everolimus, an mTOR (membrane target of rapamycin) inhibitor, significantly reduced intracellular M. bovis BCG infection alone and in conjunction with L-GSH supplementation. Furthermore, we found L-GSH supplementation coupled with in vitro everolimus treatment produced a greater effect in inhibiting the growth of intracellular Mycobacterium bovis BCG, than with the everolimus treatment alone. We also demonstrated the functions of L-GSH along with in vitro everolimus treatment in modulating the levels of cytokines such as IFN-γ, TNF-α, and IL-2 and IL-6, in favor of improving control of the mycobacterial infection. In summary, in vitro everolimus-treatment alone and in combination with oral L-GSH supplementation for three months in individuals with T2DM, was able to increase the levels of T-helper type 1 (Th1) cytokines IFN-γ, TNF-α, and IL-2 as well as enhance the abilities of granulomas from individuals with T2DM to improve control of a mycobacterial infection.

Effects of Glutathione Diminishment on the Immune Responses against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), continues to be a global health burden. We have reported that patients with marked deficiency in the production of glutathione (GSH) had impaired granulomatous effector responses against M. tb infection, which were restored when supplementing patients with liposomal GSH (lGSH). However, the effects of GSH deficiency in the lung parenchyma in altering granuloma formation and effector responses against M. tb infection remain unexplored. We aim to elucidate the effects of diethyl maleate (DEM)-induced GSH deficiency during an active M. tb infection in an in vivo mouse model. We assessed for total and reduced GSH levels, malondialdehyde (MDA) levels, cytokine profiles, granuloma formation and M. tb burden. DEM administration significantly diminished total and reduced GSH levels in the lungs and plasma and increased MDA levels in infected mice compared to sham-treated controls. DEM treatment was also associated with an increase in IL-6, TNF-α and ill-formed granulomas in infected mice. Furthermore, M. tb survival was significantly increased along with a higher pulmonary and extrapulmonary bacterial load following DEM treatment. Overall, GSH deficiency led to increased oxidative stress, impaired granuloma response, and increased M. tb survival in infected mice. These findings can provide insight into how GSH deficiency can interfere with the control of M. tb infection and avenues for novel therapeutic approaches.

General Overview of Nontuberculous Mycobacteria Opportunistic Pathogens: Mycobacterium avium and Mycobacterium abscessus.

Nontuberculous mycobacteria (NTM) are emerging human pathogens, causing a wide range of clinical diseases affecting individuals who are immunocompromised and who have underlying health conditions. NTM are ubiquitous in the environment, with certain species causing opportunistic infection in humans, including Mycobacterium avium and Mycobacterium abscessus. The incidence and prevalence of NTM infections are rising globally, especially in developed countries with declining incidence rates of M. tuberculosis infection. Mycobacterium avium, a slow-growing mycobacterium, is associated with Mycobacterium avium complex (MAC) infections that can cause chronic pulmonary disease, disseminated disease, as well as lymphadenitis. M. abscessus infections are considered one of the most antibiotic-resistant mycobacteria and are associated with pulmonary disease, especially cystic fibrosis, as well as contaminated traumatic skin wounds, postsurgical soft tissue infections, and healthcare-associated infections (HAI). Clinical manifestations of diseases depend on the interaction of the host's immune response and the specific mycobacterial species. This review will give a general overview of the general characteristics, vulnerable populations most at risk, pathogenesis, treatment, and prevention for infections caused by Mycobacterium avium, in the context of MAC, and M. abscessus.

Antimycobacterial Effects of Everolimus in a Human Granuloma Model.

Mycobacterium tuberculosis (M. tb) has been historically and is currently a threat to global public health. First-line antibiotics have been effective but proven to be burdensome as they have many potential adverse side effects. There has been a recent increase in the number of active tuberculosis (TB) cases due to a prevalence of multidrug and extensively drug-resistant strains of M. tb, and an increasing number of highly susceptible people such as those with Type 2 Diabetes (T2DM) and human immunodeficiency virus (HIV) infection. Multidrug-resistant M. tb infection (MDR-TB) is challenging to treat with existing therapeutics, so novel therapeutics and treatment strategies must be developed. Host-Directed Therapy (HDT) has been a potential target mechanism for effective clearance of infection. Host cell autophagy plays an essential role in antibacterial defense. The mammalian target of rapamycin (mTOR) has been negatively correlated with autophagy induction. Everolimus is an mTOR inhibitor that induces autophagy, but with higher water solubility. Therefore, targeting the mTOR pathway has the potential to develop novel and more effective combination drug therapy for TB. This study tested the effect of everolimus, alone and in combination with current first-line antibiotics (isoniazid and pyrazinamide), on the inhibition of M. tb inside in vitro human granulomas. We found that M. tb-infected in vitro granulomas treated with everolimus alone resulted in significantly decreased M. tb burden compared to similar granulomas in the control group. Cells treated with everolimus doses of either 1 nM or 2 nM in conjunction with pyrazinamide (PZA) produced a significant reduction in intracellular M. tb burden. Treatment groups that received everolimus alone in either 1 nM or 2 nM doses experienced a significant reduction in oxidative stress. Additionally, samples treated with 2 nM everolimus alone were observed to have significantly higher levels of autophagy and mTOR inhibition as well. Results from this study indicate that everolimus is efficacious in controlling M. tb infection in the granulomas and has additive effects when combined with the anti-TB drugs, isoniazid and pyrazinamide. This study has shown that everolimus is a promising host-directed therapeutic in the context of in vitro granuloma M. tb infection. Further study is warranted to better characterize these effects.

Orofacial rehabilitation with zygomatic implants: CAD-CAM bar and magnets for patients with nasal cancer after rhinectomy and partial maxillectomy.

Nasal carcinoma extending into the premaxilla requires radical surgical excision including rhinectomy and partial maxillectomy. Rehabilitation is complex and involves the use of removable prostheses. Three patients treated with zygomatic implants and custom-milled bars to retain an obturator and nasal prosthesis are presented.

Global gene expression profiling in mouse plasma cell tumor precursor and bystander cells reveals potential intervention targets for plasma cell neoplasia.

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eµ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.

Gene expression profiling reveals different pathways related to Abl and other genes that cooperate with c-Myc in a model of plasma cell neoplasia.

BACKGROUND: To elucidate the genes involved in the neoplastic transformation of B cells, global gene expression profiles were generated using Affymetrix U74Av2 microarrays, containing 12,488 genes, for four different groups of mouse B-cell lymphomas and six subtypes of pristane-induced mouse plasma cell tumors, three of which developed much earlier than the others. RESULTS: Unsupervised hierarchical cluster analysis exhibited two main sub-clusters of samples: a B-cell lymphoma cluster and a plasma cell tumor cluster with subclusters reflecting mechanism of induction. This report represents the first step in using global gene expression to investigate molecular signatures related to the role of cooperating oncogenes in a model of Myc-induced carcinogenesis. Within a single subgroup, e.g., ABPCs, plasma cell tumors that contained typical T(12;15) chromosomal translocations did not display gene expression patterns distinct from those with variant T(6;15) translocations, in which the breakpoint was in the Pvt-1 locus, 230 kb 3' of c-Myc, suggesting that c-Myc activation was the initiating factor in both. When integrated with previously published Affymetrix array data from human multiple myelomas, the IL-6-transgenic subset of mouse plasma cell tumors clustered more closely with MM1 subsets of human myelomas, slow-appearing plasma cell tumors clustered together with MM2, while plasma cell tumors accelerated by v-Abl clustered with the more aggressive MM3-MM4 myeloma subsets. Slow-appearing plasma cell tumors expressed Socs1 and Socs2 but v-Abl-accelerated plasma cell tumors expressed 4-5 times as much. Both v-Abl-accelerated and non-v-Abl-associated tumors exhibited phosphorylated STAT 1 and 3, but only v-Abl-accelerated plasma cell tumors lost viability and STAT 1 and 3 phosphorylation when cultured in the presence of the v-Abl kinase inhibitor, STI-571. These data suggest that the Jak/Stat pathway was critical in the transformation acceleration by v-Abl and that v-Abl activity remained essential throughout the life of the tumors, not just in their acceleration. A different pathway appears to predominate in the more slowly arising plasma cell tumors. CONCLUSION: Gene expression profiling differentiates not only B-cell lymphomas from plasma cell tumors but also distinguishes slow from accelerated plasma cell tumors. These data and those obtained from the sensitivity of v-Abl-accelerated plasma cell tumors and their phosphorylated STAT proteins indicate that these similar tumors utilize different signaling pathways but share a common initiating genetic lesion, a c-Myc-activating chromosome translocation.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

BACKGROUND: Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. RESULTS: The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. CONCLUSION: RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Insertion of c-Myc into Igh induces B-cell and plasma-cell neoplasms in mice.

We used gene targeting in mice to insert a His(6)-tagged mouse c-Myc cDNA, Myc(His), head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Emu. The insertion of Myc(His) mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, Myc(His) transgenic mice developed B-cell and plasma neoplasms, such as IgM(+) lymphoblastic B-cell lymphomas, Bcl-6(+) diffuse large B-cell lymphomas, and CD138(+) plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed Myc(His), and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of Myc(His), suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19(Arf)-Mdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that Myc(His) insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Myc-activating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection.

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.

A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection.

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis.