Researchers have sought therapeutic applications for monoclonal antibodies since their development in 1975. However, murine-derived monoclonal antibodies may cause an immunogenic response in human patients, reducing their therapeutic efficacy. Chimeric and humanized antibodies have been developed that are less likely to provoke an immune reaction in human patients than are murine-derived antibodies. Antibody fragments, bispecific antibodies, and antibodies produced through the use of phage display systems and genetically modified plants and animals may aid researchers in developing new uses for monoclonal antibodies in the treatment of disease. Monoclonal antibodies may have a number of promising potential therapeutic applications in the treatment of asthma, autoimmune diseases, cancer, poisoning, septicemia, substance abuse, viral infections, and other diseases.

Our research has focused on the ecology of commensal populations of big brown bats (Eptesicus fuscus) in Fort Collins, Colorado (USA), in relation to rabies virus (RV) transmission. We captured 35 big brown bats (Eptesicus fuscus) in late summer 2001 and held them captive for 4.8 mo. The bats were initially placed in an indoor cage for 1 mo then segregated into groups of two to six per cage. Two of the bats succumbed to rabies virus (RV) within the first month of capture. Despite group housing, all of the remaining bats were healthy over the course of the investigation; none developed rabies, although one of the rabid bats was observed to bite her cage mates. Reverse transcription-polymerase chain reaction (RT-PCR) and Taqman real-time PCR analysis of the RNA derived from the brain tissue, salivary glands, and oral swab samples confirmed RV infection in the dead bats. Rabies virus was also isolated from the brain tissue upon passage in mouse neuroblastoma cells. Nucleotide sequence analysis of the RV nucleoprotein (N) gene showed 100% identity with the N gene sequence of a 1985 E. fuscus isolate from El Paso County, Colorado. Bat sera obtained six times throughout the study were assayed for RV neutralizing antibodies using the rapid fluorescent focus inhibition test. The RV neutralizing activity in the serum was associated with the IgG component, which was purified by binding to protein G Sepharose. Five bats were RV seropositive prior to their capture and maintained titers throughout captivity. Two adult bats seroconverted during captivity. Two volant juvenile bats had detectable RV antibody titers at the first serum collection but were negative thereafter. Four seronegative bats responded to a RV vaccine administration with high titers of RV antibodies. A serologic survey of big brown bats in the roost from which one of the captive rabid bats had originated showed a significant rise in seroprevalence during 2002.

We have used the reverse transcriptase-polymerase chain reaction technique to gain insight into the pathogenesis of encephalitis caused by Borna disease virus (BDV). RNA specific for BDV was first detected in the olfactory bulb of intranasally infected rats at 6 days postinfection (p.i.). At 14 days p.i., high levels of BDV RNA were found in all brain regions, and at 26 days p.i., BDV-specific RNA was also present in the eye, nasal mucosa, and facial skin. In the chronic phase of the disease, BDV RNA was identffied in many peripheral organs but not in blood. Analysis of brain tissue for the presence of cytokine mRNAs revealed that the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and IL-lao had increased sharply at 14 and 26 days p.i. These cytokine mRNAs reached maximum levels at the peak of inflammatory reactions and decreased drastically in the chronic phase of the disease. Although IL-2 mRNA was also found in normal brain, it was markedly increased in BDV-infected brain at 14 days p.i. Expression of gamma interferon (IFN-y) mRNA, which was not observed in normal rat brain, was detected at 14 days p.i. and reached a maximum level at 38 days p.i. IL-2 and IFN-y mRNA expression correlated with expression of CD4 and CD8 mRNAs, indicating that both CD4+ and CD8+ T lymphocytes are induced in the early stages of BDV infection. Since IFN-,y and CD8 mRNA levels were still highly elevated in the chronic phase of Borna disease, it is likely that CD8+ T lymphocytes act to reduce inflammation and to ameliorate neurological signs during the chronic phase of infection.

Sera from 295 horses in the USA were examined by an indirect immunofluorescence assay and Western blot assays to determine the prevalence of Borna disease virus infection. Eight (2.7 per cent) of the samples were positive in both assays, and 18 (6.1 per cent) were positive only in the Western blot assay. The indirect fluorescence titres ranged from 1:20 to 1:80 of antibodies recognising the virus-specific antigen from Borna disease virus-infected cells. The purified virus-specific proteins isolated from infected rat brains were recognised by positive equine serum samples after immunostaining by a Western blot technique. Information obtained from the owners about the history of the seropositive horses revealed that they were either clinically normal or had a pathological diagnosis of disease unrelated to Borna disease. This is the first report of the detection of antibodies to Borna disease virus in horses in the USA. The disease may be more widespread in a subclinical form, with very long incubation periods, and may not necessarily be restricted to historically endemic areas.

Molecular epidemiological studies have linked many cryptic human rabies cases in the United States with exposure to rabies virus (RV) variants associated with insectivorous bats. In Colorado, bats accounted for 98% of all reported animal rabies cases between 1977 and 1996. The genetic divergence of RV was investigated in bat and terrestrial animal specimens that were submitted for rabies diagnosis to the Colorado Department of Public Health and Environment (CDPHE), Colorado, USA. RV isolates from animal specimens across the United States were also included in the analysis. Phylogenetic analyses were performed on partial nucleoprotein (N) gene sequences, which revealed seven principal clades. RV associated with the colonial big brown bat, Eptesicus fuscus, an bats of the genus Myotis were found to segregate into two distinct clades (I and IV). Clade I was harbored by E. fuscus and Myotis species, but was also identified in terrestrial animals such as domestic cats and striped skunks (Mephitis mephitis). Clade IV was divided into subclades IVA, IVB, and IVC; IVA was identified in E. fuscus, and Myotis species bats, and also in a fox; subclades IVB and IVC circulated predominantly in E. fuscus. Clade II was formed by big free-tailed bat (Nyctinomops macrotis) and striped skunk (Mephitis mephitis) samples. Clade III included RVs that are maintained by generally solitary, migratory bats such as the silver-haired bat (Lasionycteris noctivagans) and bats of the genus Lasiurus. Big brown bats were found to harbor this RV variant. None of the Colorado specimens segregated with clades V and VII that harbor RVs associated with terrestrial animals. Different species of bats had the same RV variant, indicating active inter-species rabies transmission. In Colorado, animal rabies occurs principally in bats, and the identification of bat RVs in cat, gray fox Urocyon cinereoargenteus), and striped skunks demonstrated the importance of rabies spillover from bats to domestic and terrestrial wildlife species.

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Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no virus-specific RNA between 6 and 30 h p.i., indicating that virus invaded the nerve ending directly, without prior replication in the muscle. At 36 h p.i., viral RNA was detected again in the masseter muscle. Selective amplification of plusand minus-strand RNA isolated from the masseter muscle at 96 h p.i. revealed that the majority of the rabies virus-specific RNA was in the positive sense, suggesting virus replication in muscle tissue during late stages of infection.

To determine whether the enhanced expression of transforming growth factor alpha (TGF alpha) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGF alpha cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGF alpha mRNA expression by northern blot analysis, TGF alpha secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGF alpha-transfected NOG-8 clones expressed high levels of a specific TGF alpha mRNA, secreted elevated levels of TGF alpha into the culture medium (177-595 ng/10(8) cells/48 h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGF alpha peptide was able to inhibit colony formation in soft agar by TGF alpha-transfected NOG-8 clones that produced high levels by TGF alpha. This inhibition suggested that TGF alpha acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGF alpha, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGF alpha cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.