Initial genetic characterization of the 1918 "Spanish" influenza virus.

The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.

Pandemic influenza--including a risk assessment of H5N1.

Influenza pandemics and epidemics have apparently occurred since at least the Middle Ages. When pandemics appear, 50% or more of an affected population can be infected in a single year, and the number of deaths caused by influenza can dramatically exceed what is normally expected. Since 1500, there appear to have been 13 or more influenza pandemics. In the past 120 years there were undoubted pandemics in 1889, 1918, 1957, 1968, and 1977. Although most experts believe we will face another influenza pandemic, it is impossible to predict when it will appear, where it will originate, or how severe it will be. Nor is there agreement about the subtype of influenza virus most likely to cause the next pandemic. The continuing spread of H5N1 highly pathogenic avian influenza viruses has heightened interest in pandemic prediction. Despite uncertainties in the historical record of the pre-virology era, study of previous pandemics may help guide future pandemic planning and lead to a better understanding of the complex ecobiology underlying the formation of pandemic strains of influenza A viruses.

Loss of functional beta 2-microglobulin in metastatic melanomas from five patients receiving immunotherapy.

BACKGROUND: In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8+ T cells) can cause the regression of even large tumors. These antitumor CD8+ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the alpha chain and a noncovalently associated, soluble protein called beta2-microglobulin (beta 2m). Loss of beta 2m generally eliminates antigen recognition by antitumor CD8+ T cells. PURPOSE: We studied the loss of beta 2m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. METHODS: We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 40 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. RESULTS: After one melanoma cell line (1074mel) was found not to express functional beta 2m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of alpha-chain mRNA but variable levels of beta 2m mRNA. In addition, no immunoreactive beta 2m protein was detected by western blot analysis. When human beta 2m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be beta 2m positive. This result was consistent with the hypothesis that loss of beta 2m resulted from immunotherapy. CONCLUSIONS: These data suggest that the loss of beta 2m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.

The virulence of the 1918 pandemic influenza virus: unraveling the enigma.

The 1918 influenza pandemic caused acute illness in 25-30% of the world's population and resulted in the death of up to 40 million people. Using lung tissue of 1918 influenza victims, the complete genomic sequence of the 1918 influenza virus is being deduced. Neither the 1918 hemagglutinin nor neuraminidase genes possess mutations known to increase tissue tropicity that account for virulence of other influenza virus strains, such as A/WSN/33 or the highly pathogenic avian influenza H5 or H7 viruses. Using reverse genetics approaches, influenza virus constructs containing the 1918 hemagglutinin and neuraminidase on an A/WSN/33 virus background were lethal in mice. The genotypic basis of this virulence has not yet been elucidated. The complete sequence of the non-structural (NS) gene segment of the 1918 virus was deduced and also tested to determine the validity of the hypothesis that enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. Results from these experiments suggest that in human cells the 1918 NS1 is a very effective interferon antagonist. Sequence analysis of the 1918 influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help elucidate how pandemic influenza virus strains emerge and what genetic features contribute to virulence in humans.

Augmentation of pre- and post-thymic T-lymphocyte responses in thymic epithelial cell grafted nude mice.

Thymic epithelial (T-E) cell cultures from newborn mice, grown on dextran beads coated with denatured collagen, were injected subcutaneously into athymic "nude" mice. After 8 weeks, enhanced in vitro migration of T-E cell grafted nude mouse bone marrow cells occurred to thymus supernatants prepared from neonatal thymus cultures. Percent migration was equal to the migration of control AKR bone marrow. In vivo T-lymphocyte responses were also significantly enhanced in T-E cell grafted nude mice as compared to controls.

The persistence of human peripheral lymphocytes, tumor infiltrating lymphocytes, and colon adenocarcinomas in immunodeficient mice.

The reconstitution of severely immunodeficient mice with human peripheral blood mononuclear cells (PBMCs) may represent a unique model system to evaluate human antitumor responses. To evaluate this possibility, we studied human PBMC reconstitution, human tumor infiltrating lymphocyte (TIL) persistence, and human colon adenocarcinoma propagation in beige/nude/xid (BNX) and in severe combined immunodeficient (SCID) mice. To evaluate human PBMC reconstitution, 75 mice received 1 x 10(7)-1 x 10(9) human PBMCs i.p. or i.v. and were studied at intervals ranging from 1 to 8 weeks by fluorescence-activated cell sorting (FACS) analysis and by measurement of circulating human immunoglobulin levels. By FACS analyses, only one of 75 mice had evidence of human PBMC persistence at 2 weeks in the spleen. Moreover, liver and peritoneum showed evidence of human cells in only 13 of 56 and 16 of 55 mice, respectively. In these mice, human cells comprised 1-77% of total cells recovered. Human immunoglobulin levels in mouse serum ranged from 0 to 34,000 micrograms/ml and correlated only weakly with evidence of human PBMC reconstitution in peripheral organs, but were generally higher in SCID mice than in BNX mice. Human TIL persistence was evaluated in BNX and SCID mice that were given 3 x 10(7) TILs i.v. (in divided doses) or 1 x 10(8) i.p. TILs along with interleukin-2 administration. At 1, 2, 7, and 14 days following TIL delivery, evidence of human TIL persistence in liver, lung, peritoneum, and spleen was evaluated by FACS analysis. Fresh organ suspensions did not contain human TILs. In mice given cyclophosphamide followed by human TILs i.p., the TILs were demonstrated at 7 days in the SCID peritoneum (leu 4 = 4%) and at 2 days in the SCID spleen (leu 4 = 2%). In BNX mice, 12 of 14 fresh human colon adenocarcinomas were propagated successfully at subcutaneous sites with latency periods ranging from 1 to 13 weeks. Enzymatic disaggregation of tumors greater than 1 cm following one passage yielded 6.5-47 x 10(6) cells with viabilities ranging from 13 to 85%. We conclude that limitations and variability exist in the use of BNX and SCID mice for human PBMC reconstitution, TIL persistence, and propagation of human colon adenocarcinomas.

Experimenting on the past: the enigma of von Economo's encephalitis lethargica.

Encephalitis lethargica (EL) was a complex and mysterious disease that appeared around the same time as the great influenza pandemic of 1918. The contemporaneous relationship of the 2 diseases led to speculation that they were causally related. Contemporary and subsequent observers conjectured that the influenza virus, directly responsible for the deaths of more than 20 million people, might also have been the cause of EL. A review of the extensive literature by observers of the EL epidemic suggests that most contemporary clinicians, epidemiologists, and pathologists rejected the theory that the 1918 influenza virus was directly responsible for EL. Disappearance of the acute form of EL during the 1920s has precluded direct study of this entity. However, modern molecular biology techniques have made it possible to examine archival tissue samples from victims of the 1918 pandemic in order to detect and study the genetic structure of the killer virus. Similarly, tissue samples from EL victims can now be examined for evidence of infection by the 1918 influenza virus.

Influenza RNA not detected in archival brain tissues from acute encephalitis lethargica cases or in postencephalitic Parkinson cases.

Encephalitis lethargica (EL) was a mysterious epidemic. temporally associated with the 1918 Spanish influenza pandemic. Numerous symptoms characterized this disease, including headache, diplopia, fever, fatal coma, delirium, oculogyric crisis, lethargy, catatonia, and psychiatric symptoms. Many patients who initially recovered subsequently developed profound, chronic parkinsonism. The etiologic association of influenza with EL is controversial. Five acute EL autopsies and more than 70 postencephalitic parkinsonian autopsies were available in the Armed Forces Institute of Pathology (AFIP) tissue repository. Two of these 5 acute EL cases had histopathologic changes consistent with that diagnosis. The remaining 3 cases were classified as possible acute EL cases as the autopsy material was insufficient for detailed histopathologic examination. RNA lysates were prepared from 29 CNS autopsy tissue blocks from the 5 acute cases and 9 lysates from blocks containing substantia nigra from 2 postencephalitic cases. RNA recovery was assessed by amplification of beta-2-microglobulin mRNA and 65% of the tissue blocks contained amplifiable RNA. Reverse transcription-polymerase chain reaction (RT-PCR) for influenza matrix and nucleoprotein genes was negative in all cases. Thus, it is unlikely that the 1918 influenza virus was neurotropic and directly responsible for the outbreak of EL.

Thyrotropin-secreting pituitary carcinoma.

Pituitary tumors rarely metastasize outside the central nervous system. Of the more than 100 reported TSH-secreting adenomas, we now describe the first carcinoma. A 40-yr-old woman had transsphenoidal surgery for a large TSH-secreting pituitary adenoma in 1984. She had increased thyroid hormone levels with a TSH that varied from 16-31 microU/mL, and an unusually high alpha-subunit that ranged from 125-150 ng/mL. Because of residual tumor, she had a left craniotomy in 1985 followed by radiation. Despite these therapies, she had a residual tumor that remained stable until January 1989 when her tumor nearly doubled in size. She received radiation therapy and octreotide with marked diminution of the tumor and clinical improvement. In August 1989, she presented with leg weakness, and magnetic resonance imaging revealed a large sacral mass. A biopsy confirmed that the sacral mass was a metastasis from the pituitary tumor. Due to additional metastases in the lung, she received 5-fluorouracil, cytoxan, and adriamycin, with marked decrease in her lesions. Further substantiation of the metastatic pituitary tumor was made when the patient returned in December 1989 with a pleural effusion containing pituitary tumor cells. Of all the reported cases of TSH-secreting adenomas, this case had the highest alpha-subunit portending future metastases. Furthermore, the apparent response to octreotide and response to chemotherapy are encouraging and suggest that new therapies should be explored. Finally, since TSH-secreting adenomas tend to be more invasive than other pituitary tumors, this case underscores the need for early diagnosis and aggressive treatment of these tumors.

The 1918 Spanish influenza: integrating history and biology.

In 1918 an influenza pandemic killed 40 million people. It is now possible to study the genetic features of the 1918 virus. Such analyses will try to answer questions about the origin and the unusual virulence of this pandemic virus.

Enhanced in vitro bone marrow cell migration and T-lymphocyte responses in aged mice given subcutaneous thymic epithelial cell grafts.

Thymic epithelial cell cultures from newborn CBA/J mice were developed and grown on collagen-coated dextran beads. These thymic epithelial cells were transplanted subcutaneously into aged syngeneic mice. After several weeks, enhanced in vitro migration of host bone marrow cells occurred to supernatants prepared from neonatal thymus cultures in thymic epithelial cell grafted mice compared to control aged mice. Percent migration equaled that of migration from control young adult mice. In addition, enhanced T-lymphocyte responses were observed in aged mice given thymic epithelial cell grafts as compared to aged controls.

Phylogenetically important regions of the influenza A H1 hemagglutinin protein.

A parsimony approach was used to construct phylogenetic trees of the H1, H2 and H3 influenza hemagglutinin subtypes. The parsimony trees were then compared with randomly generated trees to identify regions of the proteins containing the most phylogenetic information, i.e. those regions making the parsimony trees shorter. We reasoned that any areas of the hemagglutinin protein that were phylogenetically 'information-rich' would be good candidates for sites involved in virus-host interactions and their identification might lead to a better understanding of the protein. Molecular modelling, based upon the crystal structure of the H3 hemagglutinin, demonstrated that most phylogenetically important regions of the H1 subtype were on the surface of the hemagglutinin trimer, primarily in the globular region. Many corresponded to known antigenic or receptor binding sites, while others appear to be novel and specific for H1.

A thyroid biopsy with histologic features of both Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis.

We describe a 36-year-old woman with clinical, laboratory, and histologic features of both Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis. Features of the former included a hard, fixed thyroid mass and extensive involvement of perithyroidal tissues by dense fibrosis with lymphocytes, histiocytes, and plasma cells. Features supporting Hashimoto's thyroiditis included high serum titers of antimicrosomal and antithyroglobulin antibodies and the histologic findings within the thyroid gland itself: dense fibrous bands dividing the thyroid parenchyma into nodules composed of lymphoid follicles with germinal centers, plasma cells, and oxyphilic metaplasia of follicular epithelial cells. Although Riedel's thyroiditis and the fibrosing variant of Hashimoto's thyroiditis were once considered morphologic variants of the same disease, since the 1970s these diseases have been considered as distinct clinicopathologic entities. The coexistence of both diseases in a patient is rare and is probably coincidental in this instance.

Squamous cell papillomas of the esophagus: a study of 23 lesions for human papillomavirus by in situ hybridization and the polymerase chain reaction.

This study assessed squamous cell papillomas of the human esophagus for the presence of human papillomavirus (HPV) and correlated the results with histological features. Twenty-three lesions obtained by endoscopic biopsy from 17 patients were studied, first by in situ hybridization (ISH) for HPV types 6-11, 16-18, 18, and 31-33-51, and second by the polymerase chain reaction (PCR) with amplification of multiple HPV types and demonstration of amplified product by ethidium bromide staining and Southern blot hybridization for HPV types 6-11, 16, and 18 in each case. Evidence of HPV DNA was found in only one lesion, which showed HPV type 6-11 by ISH and HPV positivity by Southern blotting of the amplified product after the PCR. This case exhibited histological features suggestive of HPV infection, although no morphological changes specific to the lesion were identified. The remaining 22 lesions, including those from cases in which multiple papillomas were present, were negative for HPV. The results show that HPV DNA is frequently not detectable in esophageal squamous cell papillomas, even when highly sensitive techniques are used. These findings are consistent with the hypothesis that other pathogenetic mechanisms, such as mucosal injury and repair, are important in the etiology of these lesions.

Pulmonary metastatic disease in ameloblastoma.

Ameloblastoma is a rare disease of odontogenic origin with indeterminate metastatic potential. The first site of metastatic disease is usually the lung. We report aggressive surgical treatment of a patient with bilateral disease with five subsequent recurrences. A review of the literature suggests that in the absence of effective chemotherapy or radiation, surgery should be considered the treatment of choice for metastatic ameloblastoma confined to the lung.

Histologically discordant lymphomas with B-cell and T-cell components.

We describe the clinical, histologic, immunophenotypic, and genotypic features of five cases of histologically discordant lymphomas with B-cell and T-cell components. Three patients presented with B-cell lymphoma; T-cell lymphoma subsequently developed. One patient presented with T-cell lymphoma; B-cell lymphoma subsequently developed. One patient presented with synchronous B-cell and T-cell lymphomas. There were three men and two women. The median age at the initial diagnosis of lymphoma was 66 years. The mean interval between the development of the two lymphomas was 83 months. All patients died of disease. The mean survival was 96 months after the initial diagnosis of lymphoma and 14 months after the diagnosis of the histologically discordant lymphoma. Epstein-Barr virus was found in two cases--the B-cell lymphoma in the patient who presented with synchronous lymphomas, and the subsequent T-cell lymphoma in one of the patients who presented with B-cell lymphoma. Based on the results of immunophenotypic and genotypic analyses, these cases likely represent the occurrence of two distinct lymphoid neoplasms rather than histologic progression of the same neoplastic clone. Furthermore, a subset of these cases are Epstein-Barr virus-associated.

Effective regional therapy of experimental cancer with paralesional administration of tumour necrosis factor-alpha + interferon-gamma.

Systemically administered tumour necrosis factor (TNF) has anti-tumour effects in animal tumour models but its clinical application is limited by severe toxicity. Interferon-gamma(IFN-gamma) has been shown to augment the anti-tumour effect of TNF. We evaluated the effect of paralesional (p.I.) injections of TNF plus IFN-gamma in a murine tumour model and compared the toxicity and anti-tumour effect with that seen with systemic administration. C57BL6 mice with 10-day subcutaneous MCA sarcomas were treated with daily p.I. injections of recombinant huTNF +/- IFN-gamma for 5 days. Optimal mean survival and 30-day cure rate was seen with doses of 5 micrograms TNF-alpha + 5000 U IFN-gamma (P < 0.05 vs. control or IFN-gamma alone). Tumour response after a single i.v. injection of 0-15 micrograms TNF + 5000 U IFN-gamma was then compared with five daily p.I. injections of the same dose of TNF-alpha and IFN-gamma. All animals with p.I. injections of > 5 micrograms TNF had initial complete necrosis of tumour with a variable degree of surrounding tissue necrosis, with rapid regrowth of tumour seen in some animals. Although treatment-related mortality was similar between i.v. and p.I. therapy, there was a higher percentage of animals cured with p.I. injections with overall cure rates in treated animals at 30 days of 17% vs. 72% (i.v. vs. p.I., P < 0.01) and 13% vs. 67% (P < 0.04) in a repeat study. 2+ clinical applications.

Diagnosis of influenza virus: coming to grips with the molecular era.

Influenza viruses continually circulate and cause yearly epidemics, which kill 20,000 people in an average year in the United States. Occasionally and unpredictably, pandemic influenza strains sweep the world, infecting 20% to 40% of the world's population in a single year. In 1918, the worst influenza pandemic on record caused 675,000 deaths in the United States and up to 40 million deaths worldwide. Despite the prevalence of this virus, molecular assays for influenza diagnosis, surveillance, vaccine strain selection, and research have lagged behind such assays for other common viral pathogens. The extreme genetic variability of influenza viruses makes the design of useful molecular-based assays challenging, but several different approaches have been successfully used. RT-PCR is effective for the initial diagnosis and has greater sensitivity than other available rapid assays. Molecular assays also can be used to subtype influenza isolates, and sequence analysis of hemagglutinin may assist greatly in surveillance studies and vaccine strain selection. RT-PCR for influenza also can be performed from tissue biopsy specimens for both retrospective diagnosis and research.

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.