Louis J. DeTolla

Mice Deficient in the CXCR2 Ligand, CXCL1 (KC/GRO-α), Exhibit Increased Susceptibility to Dextran Sodium Sulfate (DSS)-induced Colitis

The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC— /— and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.

Evidence that nucleocytoplasmic Olig2 translocation mediates brain-injury-induced differentiation of glial precursors to astrocytes

The mechanisms by which neural and glial progenitor cells in the adult brain respond to tissue injury are unknown. We studied the responses of these cells to stab wound injury in rats and in two transgenic mouse models in which Y/GFP is driven either by Sox2 (a neural stem cell marker) or by Tα‐1 (which marks newly born neurons). The response of neural progenitors was low in all nonneurogenic regions, and no neurogenesis occurred at the injury site. Glial progenitors expressing Olig2 and NG2 showed the greatest response. The appearance of these progenitors preceded the appearance of reactive astrocytes. Surprisingly, we found evidence of the translocation of the transcription factor Olig2 into cytoplasm in the first week after injury, a mechanism that is known to mediate the differentiation of astrocytes during brain development. Translocation of Olig2, down‐regulation of NG2, and increased glial fibrillary acidic protein expression were recapitulated in vitro after exposure of glial progenitors to serum components or bone morphogentic protein by up‐regulation of Notch‐1. The glial differentiation and Olig2 translocation could be blocked by inhibition of Notch‐1 with the γ‐secretase inhibitor DAPT. Together, these data indicate that the prompt maturation of numerous Olig2+ glial progenitors to astrocytes underlies the repair process after a traumatic injury. In contrast, neural stem cells and neuronal progenitor cells appear to play only a minor role in the injured adult CNS.

Febrile-Range Hyperthermia Augments Neutrophil Accumulation and Enhances Lung Injury in Experimental Gram-Negative Bacterial Pneumonia

We previously demonstrated that exposure to febrile-range hyperthermia (FRH) accelerates pathogen clearance and increases survival in murine experimental Klebsiella pneumoniae peritonitis. However, FRH accelerates lethal lung injury in a mouse model of pulmonary oxygen toxicity, suggesting that the lung may be particularly susceptible to injurious effects of FRH. In the present study, we tested the hypothesis that, in contrast with the salutary effect of FRH in Gram-negative peritonitis, FRH would be detrimental in multilobar Gram-negative pneumonia. Using a conscious, temperature-clamped mouse model and intratracheal inoculation with K. pneumoniae Caroli strain, we showed that FRH tended to reduce survival despite reducing the 3 day-postinoculation pulmonary pathogen burden by 400-fold. We showed that antibiotic treatment rescued the euthermic mice, but did not reduce lethality in the FRH mice. Using an intratracheal bacterial endotoxin LPS challenge model, we found that the reduced survival in FRH-treated mice was accompanied by increased pulmonary vascular endothelial injury, enhanced pulmonary accumulation of neutrophils, increased levels of IL-1β, MIP-2/CXCL213, GM-CSF, and KC/CXCL1 in the bronchoalveolar lavage fluid, and bronchiolar epithelial necrosis. These results suggest that FRH enhances innate host defense against infection, in part, by augmenting polymorphonuclear cell delivery to the site of infection. The ultimate effect of FRH is determined by the balance between accelerated pathogen clearance and collateral tissue injury, which is determined, in part, by the site of infection.

Exposure to febrile temperature upregulates expression of pyrogenic cytokines in endotoxin-challenged mice.

Fever is a phylogenetically ancient response that is associated with improved survival in acute infections. In endothermic animals, fever is induced by a set of pyrogenic cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6] that are also essential for survival in acute infections. We studied the influence of core temperature on cytokine expression using an anesthetized mouse model in which core temperature was adjusted by immersion in water baths. We showed that raising core temperature from basal (36.5-37.5 degrees C) to febrile (39.5-40 degrees C) levels increased peak plasma TNF-alpha and IL-6 levels by 4.1- and 2. 7-fold, respectively, and changed the kinetics of IL-1beta expression in response to lipopolysaccharide challenge. TNF-alpha levels were increased predominantly in liver, IL-1beta levels were higher in lung, and IL-6 levels were widely increased in multiple organs in the warmer mice. This demonstrates that the thermal component of fever may directly contribute to shaping the host response by regulating the timing, magnitude, and tissue distribution of cytokine generation during the acute-phase response.

Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice.

Background Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. Methodology / Principal Findings The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device. Conclusions/Significance DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.

Iron, ferritin, hepatitis B surface and core antigens in the livers of chinese patients with hepatocellular carcinoma

Surgically resected specimens, consisting of tumor and adjacent non‐neoplastic liver tissue, were obtained from 40 patients with primary liver cancer at Zhong Shah Hospital, Shanghai Medical University, the Peoples Republic of China, between March 1983 and July 1984. All were hepatocellular carcinomas (HCC), one being admixed with cholangiocarcinoma. The relationship of hepatitis B virus (HBV) markers with iron and ferritin was evaluated in liver tissues from patients with primary liver cancers. The serum HBsAg (Hepatitis B surface antigen) positive rate was 80.0% (32/40). Cirrhosis was observed in 97.5% (39/40). HBsAg was identified in 82.5% (33/40) of uninvolved liver, and 35.0% (14/40) of HCC tissues (P < 0.001). HBcAg (hepatitis B core antigen) was detected in 25.0% (10/40) of liver, and 7.5% (3/40) of HCC tissues (P < 0.05). Stainable iron was found in 65.0% (26/40) of unaffected livers, and 10.0% (4/40) of HCC tissues (P < 0.001). Ferritin was demonstrated in 75% (30/40) of non‐neoplastic liver, and 40% (16/40) of HCC tissues (P < 0.001). Twenty‐two of 33 HCC patients (66.7%) with HBsAg positive cells in their livers also showed stainable iron. Of 16 patients positive for ferritin in HCC cells, iron was found in only two. Iron was found in nine of ten patients with HBcAg in non‐neoplastic hepatocytes (P = 0.056); a finding compatible with the hypothesis that iron accumulates in cells replicating HBV. The other results indicate that: (1) immunohistologic ferritin in HCC is not due to increased stainable iron; (2) tumor cells may produce ferritin; (3) polyclonal antibodies to human liver ferritin react better with non‐neoplastic hepatocytes than with HCC cells; (4) the high prevalence of HBsAg and cirrhosis in HCC suggests that HBV plays a major etiologic role in hepatocarcinogenesis in China; and (5) one case of HCC is attributed to Schistosoma japonicum infestation via cirrhosis.

Low-volume jet injection for intradermal immunization in rabbits

BackgroundThis study tested a low-volume (20–30 μl/20–30 μg DNA) jet injection method for intradermal delivery of a DNA vaccine. Jet injection offers the advantages of a needle-less system, low-cost, rapid preparation of the injected DNA solution, and a simple delivery system. More than one construct can be injected simultaneously and the method may be combined with adjuvants.ResultsLow-volume jet injection targeted delivery of a DNA solution exclusively to the dermis and epidermis of rabbits. A three injection series of plasmid DNA, encoding the Hepatitis B Surface Antigen stimulated a humoral immune response in 2/5 rabbits. One rabbit developed a significant rise in antibody titer after 1 injection and one following 2 injections. There were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10–100 times higher than jet injection.ConclusionLow-volume jet injection is a simple, effective methodology for intradermal DNA immunization.

Genetic control of acquired resistance to visceral Leishmaniasis in mice

A series ofH-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 107 amastigotes ofLeishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b × B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity toL. donovani was controlled by a dominant gene at or near theIr-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at theH-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced,H-2 CR strains withH-2b,H-2a, andH-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to otherH-2 CR strains.

A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge

Concerns about infections caused by orthopoxviruses, such as variola and monkeypox viruses, drive ongoing efforts to develop novel smallpox vaccines that are both effective and safe to use in diverse populations. A subunit smallpox vaccine comprising vaccinia virus membrane proteins A33, B5, L1, A27 and aluminum hydroxide (alum) ± CpG was administered to non-human primates, which were subsequently challenged with a lethal intravenous dose of monkeypox virus. Alum adjuvanted vaccines provided only partial protection but the addition of CpG provided full protection that was associated with a more homogeneous antibody response and stronger IgG1 responses. These results indicate that it is feasible to develop a highly effective subunit vaccine against orthopoxvirus infections as a safer alternative to live vaccinia virus vaccination.

Disseminated Simian Varicella Virus Infection in an Irradiated Rhesus Macaque (Macaca mulatta)

ABSTRACT We describe correlative clinicopathological/virological findings from a simian varicella virus (SVV)-seronegative monkey that developed disseminated varicella 105 days after gamma-irradiation. Twelve other monkeys in the colony were also irradiated, none of which developed varicella. Before irradiation, sera from the monkey that developed disseminated infection and one asymptomatic monkey were available. Analysis indicated that subclinical reactivation of latent SVV from an asymptomatic irradiated monkey likely led to disseminated varicella in the seronegative irradiated monkey. These findings parallel those from humans with disseminated varicella infection and support the usefulness of SVV infection as a model for human varicella-zoster virus infection, particularly virus reactivation after gamma-irradiation.