The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation.

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.     

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.     

Role of cognitive parameters in dengue hemorrhagic fever and dengue shock syndrome

Dengue is becoming recognized as one of the most important vector-borne human diseases. It is predominant in tropical and subtropical zones but its geographical distribution is progressively expanding, making it an escalating global health problem of today. Dengue presents with spectrum of clinical manifestations, ranging from asymptomatic, undifferentiated mild fever, dengue fever (DF), to dengue hemorrhagic fever (DHF) with or without shock (DSS), a life-threatening illness characterized by plasma leakage due to increased vascular permeability. Currently, there are no antiviral modalities or vaccines available to treat and prevent dengue. Supportive care with close monitoring is the standard clinical practice. The mechanisms leading to DHF/DSS remains poorly understood. Multiple factors have been attributed to the pathological mechanism, but only a couple of these hypotheses are popular in scientific circles. The current discussion focuses on underappreciated factors, temperature, natural IgM, and endotoxin, which may be critical components playing roles in dengue pathogenesis.     

A novel method of sputum processing for cytologic diagnosis of lung cancer

OBJECTIVE: To compare the diagnostic sensitivity and cytologic findings of the sputum-processing method with those of the traditional sputum smear method. STUDY DESIGN: From May to December 2001, 300 consecutive sputum samples were collected from 168 patients suspected of having lung cancer in the Chest Department, Taipei Veterans General Hospital. After the sputum smear method, the remaining sputum material was processed by homogenization, filtration and fixation. All the slides were stained with Papanicolaou stain and reviewed by 2 cytologists. RESULTS: Of the 300 sputum samples, 141 from 79 patients were finally diagnosed as lung malignancies. The mean number of sputum samples was 1.78 per patient. Among the 79 patients, 46 had peripheral lung malignancies (58.2%). The overall diagnostic sensitivities of sputum smear and sputum-processing methods were 29% and 31%, respectively (P = .776). Tumor location and cell types did not change the difference significantly; however, among patients with small cell lung cancer, there was a higher detection rate with the sputum-processing method (50% vs. 20%, P = .350). Eight patients with negative results with the sputum smear had positive results with the sputum-processing method (8 of 79 = 10.1%). Microscopic morphologic differences between the 2 methods were described. CONCLUSION: There was no significant difference in diagnostic sensitivity between the sputum smear and sputum-processing methods. However, the sputum-processing method may play a role in patients with small cell lung carcinoma.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.