Yoshitsugu Tagawa

Identification and immunohistochemical localization of macrophage migration inhibitory factor in human cornea

We identified macrophage migration inhibitory factor (MIF) mRNA expression in human cornea, and demonstrated its immunohistological localization. Reverse transcription‐polymerase chain reaction analysis revealed that MIF mRNA was expressed in both the corneal epithelial and endothelial cells. Immunohistochemical study using the polyclonal antibody prepared from immunizing a rabbit with human recombinant MIF showed that MIF was present in the basal cells of corneal epithelium and endothelial cells. The fact that MIF exists in those cells of the cornea indicates that MIF may play an important role in corneal cell immunity and cellular differentiation.

Novel Human Adenovirus Causing Nosocomial Epidemic Keratoconjunctivitis

ABSTRACT In 2000, we encountered cases of nosocomial infections with epidemic keratoconjunctivitis (EKC) at a university hospital in Kobe, in the western part of Japan. Two human adenovirus (HAdV) strains, Kobe-H and Kobe-S, were isolated from patients with nosocomial EKC infection. They were untypeable by existing neutralizing antisera; however, the isolate was neutralized with homologous antisera. We then encountered several cases of EKC due to nosocomial infections in eye clinics in different parts of Japan. A total of 80 HAdVs were isolated from patients with EKC at eight different hospitals. The partial hexon gene sequences of the isolates were determined and compared to those of the prototype strains of 51 serotypes. All isolates had identical partial hexon nucleotide sequences. Phylogenetic analysis classified these isolates into species of HAdV-D. The isolates showed 93.9 to 96.7% nucleotide identity with HAdV-D prototype strains, while all 32 HAdV-D prototype strains ranged from 93.2 to 99.2% identity. The sequences of the loop 2 and fiber knob regions from the representative strain, Kobe-H, were dissimilar in all prototype strains of 51 serotypes. We believe that this virus is a novel serotype of HAdV that causes EKC.

Monocyte Chemotactic Protein-1 in the Vitreous of Patients with Proliferative Diabetic Retinopathy

Purpose: To investigate the correlation between monocyte chemotactic protein-1 (MCP-1) levels in the vitreous and clinical findings in eyes with proliferative diabetic retinopathy (PDR). Methods: We assayed MCP-1 levels by ELISA in vitreous samples of 88 consecutive patients with PDR (52 eyes) and macular holes or idiopathic epimacular membrane (controls, 36 eyes). Results: The level of MCP-1 in the vitreous was 2,097.5 ± 1,099.4 pg/ml (mean ± SD) in PDR, and 504.3 ± 405.6 pg/ml in the controls. In PDR eyes, multivariate regression analysis revealed a significant association between MCP-1 levels in the vitreous and the degree of proliferative membrane, and a significant negative association between MCP-1 levels and the extent of preoperative retinal photocoagulation. Conclusion: The results suggest that MCP-1 may play a role in the development of the proliferative phase of PDR.

Molecular Diagnosis of Human Adenoviruses D and E by a Phylogeny-Based Classification Method Using a Partial Hexon Sequence

ABSTRACT Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.

Analysis of the complete genome sequence of epidemic keratoconjunctivitis-related human adenovirus type 8, 19, 37 and a novel serotype.

We determined the complete genome sequence of epidemic keratoconjunctivitis (EKC)-related human adenoviruses (HAdVs). We analysed a total of 12 HAdV strains; three prototype strains and two HAdV-8, three HAdV-19 and three HAdV-37 clinical isolates from EKC patients in Japan, and one novel serotype of HAdV. Genome organization of these serotypes was identical to those of the recently determined HAdV-19 and HAdV-37. The identities of the whole genome were over 99 % among strains from the same serotype, except for HAdV-19p, which is not associated with conjunctivitis, resulting in the formation of a distinct cluster in the phylogenetic analysis. The penton, loop 1 and loop 2 of hexon, early region 3 (E3) and fiber were hypervariable regions between serotypes. Results suggest that the HAdV-19 clinical strain is a recombinant of HAdV-19p-like and HAdV-37-like strains, and that the acquisition of the penton, E3 or fiber may be related to ocular tropism.

Hepatocyte growth factor levels in the vitreous of patients with proliferative vitreoretinopathy.

PURPOSE To determine whether the hepatocyte growth factor is increased in the vitreous of patients with proliferative vitreoretinopathy. METHODS We assayed hepatocyte growth factor levels by enzyme-linked immunosorbent assay (ELISA) in vitreous samples from 65 consecutive eyes (65 patients) with proliferative vitreoretinopathy (23 eyes), rhegmatogenous retinal detachment (22 eyes), and macular hole or idiopathic epiretinal membrane (control subjects, 20 eyes) having undergone pars plana vitrectomy. RESULTS Vitreous levels of hepatocyte growth factor were 3.94 +/- 2.29 (mean +/- SD) ng/ml in proliferative vitreoretinopathy, 2.02 +/- 0.84 ng/ml in rhegmatogenous retinal detachment, and 2.16 +/- 1.39 ng/ml in the control subjects. The vitreous levels in proliferative vitreoretinopathy were much greater than levels in rhegmatogenous retinal detachment (P =.0002) and in the control subjects (P =.0007). In proliferative vitreoretinopathy, there was a tendency toward higher levels in eyes with grade D by the Retina Society terminology. CONCLUSION The results suggest the possibility that hepatocyte growth factor may play a role in the pathogenesis of proliferative vitreoretinopathy.

Epidemic Keratoconjunctivitis Due to the Novel Hexon-Chimeric-Intermediate 22,37/H8 Human Adenovirus

ABSTRACT In a 2-month period in 2003, we encountered an outbreak of epidemic keratoconjunctivitis (EKC) in Japan. We detected 67 human adenoviruses (HAdVs) by PCR from eye swabs of patients with EKC at five eye clinics in different parts of Japan. Forty-one of the 67 HAdV DNAs from the swabs were identified as HAdV-37 by phylogenetic analysis using a partial hexon gene sequence. When the restriction patterns of these viral genomes were compared with that of the HAdV-37 prototype strain, one isolate showed a never-before-seen restriction pattern. Within 1 year, we encountered three more EKC cases caused by a genetically identical virus: two nosocomial infections at two different university hospitals and a sporadic infection at an eye clinic. We determined the nucleotide sequences of the full-length hexon and fiber genes of these isolates and compared them to those of the 51 prototype strains. Surprisingly, the sequence of the hexon (ε determinant) loop-1 and -2 regions showed the highest nucleotide identity with HAdV-22, a rare EKC isolate. However, the nucleotide sequence of the fiber gene was identical to that of the HAdV-8 prototype strain. 22 We propose that this virus is a new hexon-chimeric intermediate HAdV-22,37/H8, and may be an etiological agent of EKC.

Five New Genome Types of Adenovirus Type 37 Caused Epidemic Keratoconjunctivitis in Sapporo, Japan, for More Than 10 Years

ABSTRACT Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

New Genome Type of Adenovirus Serotype 4 Caused Nosocomial Infections Associated with Epidemic Conjunctivitis in Japan

ABSTRACT Human adenovirus type 4 is one of the major serotypes isolated from patients with adenoviral conjunctivitis. In 2001 we encountered nosocomial infections with epidemic conjunctivitis in the ophthalmology ward of one hospital in Sapporo, which is in the northern part of Japan. Adenoviruses were isolated from the patients with this nosocomial infection and identified as adenovirus type 4 (AdV-4) by a neutralization test with serotype-specific antiserum. When the cleavage patterns of the isolates were compared with the full viral genome with BamHI and SmaI, the cleavage patterns of the isolates were shown to be different from those of AdV-4p and other previously known AdV-4 variants. The nucleotide sequences of the fiber gene of the isolates showed the highest homologies (94.3%) with AdV-4 among the nucleotide sequences available from GenBank and formed a monophyletic cluster along with the prototype strain of AdV-4. The isolates, however, were located in a different lineage from those of AdV-4p and the AdV-4 variant from the sporadic infections. We conclude that the nosocomial infection that appeared in 2001 was caused by a new genome type of AdV-4, which was designated AdV-4c.

Monocyte chemotactic protein-1 levels in the Vitreous of patients with proliferative vitreoretinopathy

PURPOSE To assess the potential role of monocyte chemotactic protein-1 (MCP-1) in the pathogenesis of proliferative vitreoretinopathy (PVR) and to investigate its possible interaction with the macrophage migration inhibitory factor (MIF). METHODS We assayed MCP-1 and MIF levels in the vitreous samples of 85 consecutive patients with PVR (29 eyes), rhegmatogenous retinal detachment (RRD; 22 eyes), and macular hole or idiopathic epimacular membrane (controls; 34 eyes), by enzyme-linked immunosorbent assay. RESULTS Vitreous levels of MCP-1 were 1760.7 +/- 471.3 pg/mL (mean +/- SD) in PVR patients, 1200.4 +/- 579.8 pg/mL in RRD patients, and 436.3 +/- 286.1 pg/mL in the controls. Vitreous MCP-1 levels in PVR patients were significantly higher than those in RRD patients and in the controls (P <.0001, respectively). MCP-1 levels in grade C of PVR (1883.7 +/- 479.5 pg/mL) were significantly greater than those in grade D (1437.8 +/- 258.8 pg/mL) (P =.0112). Vitreous concentrations of MCP-1 had no correlation with those of MIF. CONCLUSIONS The results indicate the possibility that MCP-1 may have a role mainly in the early stage of PVR and that the role of MCP-1 in PVR may differ from that of MIF.