Further Assessment of Monkeypox Virus Infection in Gambian Pouched Rats (Cricetomys gambianus) Using In Vivo Bioluminescent Imaging

Monkeypox is a zoonosis clinically similar to smallpox in humans. Recent evidence has shown a potential risk of increased incidence in central Africa. Despite attempts to isolate the virus from wild rodents and other small mammals, no reservoir host has been identified. In 2003, Monkeypox virus (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (Cricetomys gambianus). Therefore, we investigated the potential reservoir competence of the Gambian pouched rat for MPXV by utilizing a combination of in vivo and in vitro methods. We inoculated three animals by the intradermal route and three animals by the intranasal route, with one mock-infected control for each route. Bioluminescent imaging (BLI) was used to track replicating virus in infected animals and virological assays (e.g. real time PCR, cell culture) were used to determine viral load in blood, urine, ocular, nasal, oral, and rectal swabs. Intradermal inoculation resulted in clinical signs of monkeypox infection in two of three animals. One severely ill animal was euthanized and the other affected animal recovered. In contrast, intranasal inoculation resulted in subclinical infection in all three animals. All animals, regardless of apparent or inapparent infection, shed virus in oral and nasal secretions. Additionally, BLI identified viral replication in the skin without grossly visible lesions. These results suggest that Gambian pouched rats may play an important role in transmission of the virus to humans, as they are hunted for consumption and it is possible for MPXV-infected pouched rats to shed infectious virus without displaying overt clinical signs.     

Laboratory Investigations of African Pouched Rats (Cricetomys gambianus) as a Potential Reservoir Host Species for Monkeypox Virus

Monkeypox is a zoonotic disease endemic to central and western Africa, where it is a major public health concern. Although Monkeypox virus (MPXV) and monkeypox disease in humans have been well characterized, little is known about its natural history, or its maintenance in animal populations of sylvatic reservoir(s). In 2003, several species of rodents imported from Ghana were involved in a monkeypox outbreak in the United States with individuals of three African rodent genera (Cricetomys, Graphiurus, Funisciurus) shown to be infected with MPXV. Here, we examine the course of MPXV infection in Cricetomys gambianus (pouched Gambian rats) and this rodent species’ competence as a host for the virus. We obtained ten Gambian rats from an introduced colony in Grassy Key, Florida and infected eight of these via scarification with a challenge dose of 4X10⁴ plaque forming units (pfu) from either of the two primary clades of MPXV: Congo Basin (C-MPXV: n = 4) or West African (W-MPXV: n = 4); an additional 2 animals served as PBS controls. Viral shedding and the effect of infection on activity and physiological aspects of the animals were measured. MPXV challenged animals had significantly higher core body temperatures, reduced activity and increased weight loss than PBS controls. Viable virus was found in samples taken from animals in both experimental groups (C-MPXV and W-MPXV) between 3 and 27 days post infection (p.i.) (up to 1X10⁸ pfu/ml), with viral DNA found until day 56 p.i. The results from this work show that Cricetomys gambianus (and by inference, probably the closely related species, Cricetomys emini) can be infected with MPXV and shed viable virus particles; thus suggesting that these animals may be involved in the maintenance of MPXV in wildlife mammalian populations. More research is needed to elucidate the epidemiology of MPXV and the role of Gambian rats and other species.     

Lipid Metabolism,Oxidative Stress and Cell Death Are Regulated by PKC Delta in a Dietary Model of Nonalcoholic Steatohepatitis

Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ^−/− mice and wildtype littermates a control or MCD diet. PKCδ^−/− primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ^−/− mice fed the MCD diet. The hepatic expression of key regulators of β-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ^−/− mice and changes in serum triglyceride were blocked in PKCδ^−/− mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ^−/− mice. MCD diet-induced NADPH oxidase activity and p47^phox membrane translocation were blunted and blocked, respectively, in PKCδ^−/− mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ^−/− mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ^−/− mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.     

New Chinchillid Rodents (Hystricognathi: Caviomorpha) from Northern Chile and Bolivia Fill a 17-Million-Year Gap in the Pan-Chinchilline Fossil Record

Journal of Mammalian Evolution - The fossil record of chinchillid rodents (Hystricomorpha: Caviomorpha) begins in the early Miocene. However, nearly all remains have thus far been limited to the...     

Sex-specific HDL cholesterol changes with weight loss and their association with anthropometric variables: the LIFE study

Decrease in the level of high-density lipoprotein cholesterol (HDLC) has been observed in women who start dieting, but not in men. Patterns of HDLC change during intentional weight loss through 30-months of follow-up, and their association with changes in anthropometric measurements were examined in obese women (N = 112) and men (N = 100). Missing HDLC values at 6-, 12-, 18-, and 30-month follow-up (N = 16, 34, 55, and 50, respectively) due to dropout were imputed by multiple imputation. Mean ages and BMIs of subjects at baseline were 47.2 years and 34.8 kg/m(2) for women, and 50.4 years and 35.0 kg/m(2) for men. On average, participants lost weight steadily for 12 months, followed by slow regain. During the first 6 months, HDLC decreased significantly in women (-4.1 mg/dl, P = 0.0007), but not in men. Significant HDLC increases were observed in both men and women from 6- to 12-month follow-up. HDLC changes in women were positively associated with changes in hip circumference from baseline to 12-month independent of changes in triglycerides (TG), glucose, and insulin. Rapid decrease of predominantly subcutaneous fat in the femoral and gluteal area might be associated with HDLC decrease in women during initial weight loss.     

Cryptococcus-Related Immune Reconstitution Inflammatory Syndrome (IRIS): Pathogenesis and its Clinical Implications

This review provides an overview of Cryptococcus neoformans immunology and focuses on the pathogenesis of Cryptococcus-related paradoxical immune reconstitution inflammatory syndrome (IRIS). Cryptococcal IRIS has three phases: (1) before antiretroviral therapy (ART), with a paucity of cerebrospinal fluid (CSF) inflammation and defects in antigen clearance; (2) during initial ART immune recovery, with pro-inflammatory signaling by antigen-presenting cells without an effector response; and (3) at IRIS, a cytokine storm with a predominant type-1 helper T-cell (Th₁) interferon-gamma (IFN-γ) response. Understanding IRIS pathogenesis allows for risk stratification and customization of HIV/AIDS care. In brief, persons at high IRIS risk may benefit from enhancing microbiologic clearance by use of adjunctive agents in combination with amphotericin, prolonging initial induction therapy, and/or increasing the initial consolidation antifungal therapy dose to at least 800 mg of fluconazole daily until the 2-week CSF culture is known to be sterile. Prophylactic anti-inflammatory therapies or undue delay of ART initiation in an attempt to prevent IRIS is unwarranted and may be dangerous.     

Switching multiple sclerosis patients with breakthrough disease to second-line therapy

Background

Multiple sclerosis (MS) patients with breakthrough disease on immunomodulatory drugs are frequently offered to switch to natalizumab or immunosuppressants. The effect of natalizumab monotherapy in patients with breakthrough disease is unknown.

Methods

This is an open-label retrospective cohort study of 993 patients seen at least four times at the University of California San Francisco MS Center, 95 had breakthrough disease on first-line therapy (60 patients switched to natalizumab, 22 to immunosuppressants and 13 declined the switch [non-switchers]). We used Poisson regression adjusted for potential confounders to compare the relapse rate within and across groups before and after the switch.

Results

In the within-group analyses, the relapse rate decreased by 70% (95% CI 50,82%; p<0.001) in switchers to natalizumab and by 77% (95% CI 59,87%; p<0.001) in switchers to immunosuppressants; relapse rate in non-switchers did not decrease (6%, p = 0.87). Relative to the reduction among non-switchers, the relapse rate was reduced by 68% among natalizumab switchers (95% CI 19,87%; p = 0.017) and by 76% among the immunosuppressant switchers (95% CI 36,91%; p = 0.004).

Conclusions

Switching to natalizumab or immunosuppressants in patients with breakthrough disease is effective in reducing clinical activity of relapsing MS. The magnitude of the effect and the risk-benefit ratio should be evaluated in randomized clinical trials and prospective cohort studies.     

Sphingosine Kinase Activity Is Not Required for Tumor Cell Viability

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.     

Co-Expression of SERCA Isoforms,Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs) by reducing their apparent affinity for Ca²⁺. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca²⁺ affinity of SERCA (K _Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.     

Structure guided design of a series of sphingosine kinase (SphK) inhibitors

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other’s loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (?/?) mice.