Hematologic differences in heterophile-positive and heterophile-negative infectious mononucleosis.

Infectious mononucleosis (IM) due to all causes is characterized by atypical lymphocytosis. We sought to compare hematologic parameters of infectious mononucleosis due to Epstein-Barr virus (EBV) infection (heterophile antibody (HA) positive) with mononucleosis due to other causes. Mono-Latex Slide Agglutination Test results and complete blood counts (CBC) of 147 patients with mononucleosis were retrospectively analyzed. Leukocyte count, absolute lymphocyte count, and presence of atypical lymphocytes in EBV-positive and EBV-negative groups were statistically compared. We analyzed 68 EBV-positive and 79 EBV-negative cases. EBV-positive patients were significantly younger than EBV-negative patients were. Mean total WBC count and mean absolute lymphocyte count were significantly higher in EBV-positive patients. Absolute lymphocytosis, absolute leukocytosis, and atypical lymphocytosis were also significantly more frequent in EBV-positive patients. Leukopenia was more frequently seen in EBV-negative patients.

Persistent productive Epstein-Barr virus replication in normal epithelial cells in vivo.

Productive Epstein-Barr virus (EBV) replication characterizes hairy leukoplakia, an oral epithelial lesion typically occurring in individuals infected with human immunodeficiency virus (HIV). Serial tongue biopsy specimens were obtained from HIV-infected subjects before, during, and after valacyclovir treatment. EBV replication was detected by Southern hybridization to linear terminal EBV genome fragments, reverse-transcriptase polymerase chain reaction amplification of EBV replicative gene transcripts, immunohistochemical detection of EBV replicative protein, and in situ hybridization to EBV DNA. EBV replication was detected in both hairy leukoplakia and normal tongue tissues. Valacyclovir treatment completely abrogated EBV replication in vivo, resulting in resolution of hairy leukoplakia when it was present. EBV replication returned in normal tongue epithelial cells after valacyclovir treatment. These data suggest that normal oral epithelium supports persistent EBV infection in individuals infected with HIV and that productive EBV replication is necessary but not sufficient for the pathogenesis of oral hairy leukoplakia.

T-gamma gene rearrangement and CMV mononucleosis.

A clonal T-gamma rearrangement was found in peripheral blood and bone marrow in a 57-year-old female who presented with 6-week history of fevers, night sweats, and weight loss. Splenomegaly, hemolytic anemia, atypical lymphocytosis, a marrow lymphoid aggregate, and elevated LDH had suggested lymphoproliferative disease. However, IgM serology for cytomegalovirus (CMV) was positive. With observation alone, her clinical features improved over 4 weeks with normalization of the blood count and disappearance of CMV viremia and the aberrant T-gamma clone. Acute CMV infection may mimic lymphoproliferative disease. T-gamma gene rearrangement may be part of the immune response to CMV infection and is not specific to lymphoid neoplasia.

Composite nodular lymphocyte-predominance Hodgkin disease and gamma-heavy-chain disease: a case report and review of the literature.

The association of Hodgkin disease with monoclonal gammopathy has rarely been reported. We present a case of a 48-year-old woman with a history of autoimmune hemolytic anemia and Graves disease who presented with hepatosplenomegaly and a gamma-heavy-chain paraprotein. Histopathology of lymph node and bone marrow revealed nodular lymphocyte-predominance Hodgkin disease, while examination of the spleen revealed plasmacytosis consistent with gamma-heavy-chain disease. Following splenectomy, the patient has remained in complete remission for both conditions with no further treatment. To our knowledge, this is the first report of a patient with both gamma-heavy-chain disease and nodular lymphocyte-predominance Hodgkin disease. Given recent data indicating the B-cell nature of this form of Hodgkin disease, the authors propose that in this unique case there may be a clonal relationship between these 2 concurrent B-cell lymphoproliferative processes.

CD13-positive anaplastic large cell lymphoma of T-cell origin--a diagnostic and histogenetic problem.

The expression of myelomonocytic-associated antigens in anaplastic large cell lymphomas (ALCLs), particularly those presenting in extranodal sites, can make their distinction from extramedullary myeloid cell tumors (EMCTs) or histiocytic tumors problematic. Yet, this distinction is clinically significant because of its therapeutic and prognostic implications. Herein, we describe a case of extranodal anaplastic lymphoma kinase-positive CD30-positive ALCL of T-cell origin in a 12-year-old boy, which was initially called an EMCT because of the expression of CD13 and HLA-DR detected by flow cytometry and the absence of other T-cell-related surface markers. However, the detection of cytoplasmic CD3 by flow cytometry prompted further studies. The tumor was composed of large cells with abundant slightly eosinophilic vacuolated cytoplasm and ovoid or reniform nuclei with a few small nucleoli. Using immunohistochemistry, the tumor was positive for CD45, CD30, CD45RO, and CD43 with a strong cytoplasmic and nuclear anaplastic lymphoma kinase stain. The tumor cells showed a T-cell clonal genotype. Electron microscopy revealed no ultrastructural features of myelomonocytic or histiocytic origin. The patient responded well to the chemotherapy and was in complete remission for 10 months at the time of submission of this manuscript. Review of the literature showed inconsistencies regarding the diagnosis, nomenclature, and, therefore, treatment and prognosis of these tumors. In addition, the CD13 expression in ALCL raises some histogenetic questions and may indicate origin from a pluripotent stem cell, misprogramming during malignant transformation, or a microenvironmental effect on lymphoid cell expression of surface antigens. Therefore, ALCL should be considered in the differential diagnosis of EMCTs or histiocytic tumors, particularly when surface marker lineage assignment is ambiguous.

Acute myeloid leukemia associated with hemophagocytic syndrome and t(4;7)(q21;q36).

Hemophagocytic syndrome (HS) is a histiocytic reactive process often associated with infections and/or malignancies. Clonal karyotypic abnormalities have been the hallmark of several hematological malignancies and have been shown to be of clinical significance in terms of both diagnosis and prognosis. While there are limited reports of both clonal and nonclonal abnormalities in HS, their clinical significance has not been established. Detection of such clonal abnormalities, as seen in some cases of HS, may indicate the presence of an occult malignant process, even when there is no microscopic evidence of a hematological malignancy. We report a case of HS in a child with clonal t(4;7)(q21;q36) which later progressed to acute myeloid leukemia (AML) with further clonal evolution. Our case strengthens the argument that cytogenetic studies in HS may be important in identifying the underlying occult malignant process.

GLUT-3 expression in human skeletal muscle.

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Measurement of estrogen receptors in intact cells by flow cytometry.

BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. RESULTS: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. CONCLUSIONS: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.

Marked increase in L-selectin-negative T cells in neonatal pertussis. The lymphocytosis explained?

The detailed immunophenotype of peripheral blood lymphocytes from a neonate with pertussis was determined by flow cytometry and compared with results from cord blood from healthy newborns. Most (72%) of the lymphocytes were CD3+ T cells with a normal CD4/CD8 ratio (2.5). The T cells were largely HLA-DR negative and CD45RA+, consistent with unstimulated naïve T cells. Almost all of the CD4+ T cells were Leu8 (L-selectin, CD62L) negative, while almost all of the CD8+ T cells were CD28+. There was no increase in CD7- CD4+ T cells (Th2-like). No relative increase in CD16/56+ NK cells (5%) or CD19/20+ B cells was seen. The most dramatic finding in this case was the remarkable lack of expression of L-selectin by the T cells. L-selectin expression is associated with homing of peripheral blood lymphocytes to lymph nodes. The dramatic reduction in L-selectin expression of the T lymphocytes in pertussis, perhaps induced by pertussis toxin, likely prevents homing of the T cells to peripheral lymphoid tissues and provides a likely explanation for the marked lymphocytosis noted in this disease.

Fulminant parvovirus infection following erythropoietin treatment in a patient with acquired immunodeficiency syndrome.

We report the case of a 41-year-old black man with acquired immunodeficiency syndrome who developed a severe chronic anemia due to parvovirus infection. Bone marrow biopsy revealed erythroid aplasia. The infectious nature of the anemia was not recognized, and the patient was treated with erythropoietin. The patient's reticulocyte response was inadequate, however, and he remained anemic. A second bone marrow biopsy showed erythroid hyperplasia and prominent intranuclear parvovirus inclusions within erythroid progenitors. Erythropoietin was discontinued and was followed by a course of intravenous immunoglobulin, which resulted in rapid correction of anemia. To our knowledge, this is the first reported case of fulminant human parvovirus infection exacerbated by erythropoietin administration and documented by sequential bone marrow histologic examination. This case illustrates the critical importance of considering parvovirus in the etiology of chronic anemia with erythroid aplasia in immunocompromised patients.

Differential expression of the HHV-8 vGCR cellular homolog gene in AIDS-associated and classic Kaposi's sarcoma: potential role of HIV-1 Tat.

Human herpesvirus 8 (HHV-8) has been causally linked to Kaposi's sarcoma (KS). There is significant homology between some HHV-8 genes and cellular genes including D-type cyclin (vCYC), G protein coupled receptor (vGCR), macrophage inflammatory proteins (vMIP-I, vMIP-II), bcl-2 (vBCL2), interferon regulatory factor-1 (vIRF1), interleukin-6 (vIL6), and complement-binding protein (vCBP). In this study, we analyzed expression of these viral homologs and HIV-1 Tat by reverse-transcriptase polymerase chain reaction (RT-PCR) coupled with Southern blot hybridization in AIDS-KS (AKS) tissue, classic KS tissue(CKS), and peripheral blood mononuclear cells, and phorbol ester (TPA)-treated and untreated HHV-8 positive lymphoma cells (BCBL1). While vCYC (AKS 6 of 6; CKS 3 of 3), vMIP-I (AKS 5 of 6, CKS 3 of 3), vBCL2 (AKS 6 of 6; CKS 3 of 3), and vIRF1 (AKS 5 of 6, CKS 3 of 3) transcripts were detected in both AKS and CKS, vGCR and HIV-1 Tat were expressed only in AKS samples (vGCR: AKS 3 of 6, CKS 0 of 3; Tat: AKS 4 of 6, CKS 0 of 3). vMIPII, vCBP, and vIL6 expression were not detected in any KS samples. Since vGCR expression is limited to AKS, it is possible that vGCR is activated by HIV-1 Tat. These results suggest that HIV-1 Tat may contribute to AKS pathogenesis through the tumorigenic and angiogenic effects of vGCR.

Methylenetetrahydrofolate reductase (MTHFR): the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population.

The polymorphic mutation C677T in the gene of MTHFR is considered a risk mutation for spina bifida and vascular disease. Another common mutation on the MTHFR gene, A1298C, has also been described as another risk mutation. We studied the frequencies of these two mutations on DNA samples from healthy Jewish individuals and compared them to the frequency of these mutations in DNA samples obtained from healthy individuals in South Texas. The presence of the C677T allele was determined by PCR and Hinf I digestion, and mutation A1298C by PCR and Mbo II digestion. A total of 310 alleles was examined for C677T in the Ashkenazi samples and 400 alleles in the non-Jewish samples. The rate of C677T among the Ashkenazi Jewish alleles was 47.7% as compared to 28.7% among the alleles from the non-Jewish population. The difference is statistically significant, P < 0.0005. Mutation A1298C was examined in 298 alleles of Jewish individuals and 374 alleles of non-Jewish counterparts from Texas. The rate of the A1298C mutation in the Jewish samples was 27.2% whereas in the non-Jewish was 35%. This was also statistically significant, P < 0.031. No individuals were homozygous for both mutations or were found to be homozygous for one mutation with heterozygosity of the other mutation, and that the C677T and the A1298C alleles did not occur in cis position. This study shows a unique distribution of C677T and the A1298C alleles among the Ashkenazi Jews. In spite of high frequency of C677T mutation, spina bifida is less common among Ashkenazi Jews. Further studies are needed to establish whether the C677T and the A1298C mutations have an impact on vascular disease in the Ashkenazi Jewish population.

Monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target.

Cutaneous biopsies of five eschars and two rash lesions from five patients from New York City with documented rickettsialpox were examined by immunohistochemical methods with a monoclonal antibody directed against spotted fever group rickettsial lipopolysaccharide for the presence and cellular location of Rickettsia akari Rickettsiae were identified in all of the five patients, with good concordance of results for the same biopsy tissues with previously reported results by the direct immunofluorescence method. In contrast with immunofluorescence, which did not reveal the location of the organisms, immunohistochemical examination demonstrated R. akari to be in perivascular cells, morphologically resembling macrophages. Evaluation with double staining for rickettsiae and either CD68 or Factor VIII-related antigen revealed that the predominant infected cell type was CD68-positive macrophages, and only a rare rickettsia was detected in vascular endothelium, the major target cell for other rickettsioses. These results provide a diagnostic method for rickettsialpox and other spotted fever group rickettsioses and indicate that the elucidation of the pathogenesis of rickettsialpox must take into account that its target cell differs from that of Rocky Mountain spotted fever, boutonneuse fever, louse-borne typhus fever, and murine typhus.

Hydrocortisone activation of human herpesvirus 8 viral DNA replication and gene expression in vitro.

BACKGROUND: Patients undergoing chronic steroid therapy for organ transplantation are at increased risk for development of human herpes virus 8(HHV-8)-associated Kaposi's sarcoma (KS). It has also been reported that following steroid withdrawal, KS lesions often undergo partial or complete regression. METHODS: We have examined the effect of corticosteroid treatment on HHV-8 replication, gene expression, and lytic protein expression in BCBL-1 cells in vitro. BCBL-1 cells were collected after culture for 24-72 hr with hydrocortisone (HC) 1-5 microM, phorbol ester 20 ng/ml (positive control), and culture medium only (negative control). HHV-8 genomic conformation was examined by Gardella gel analysis. mRNA expression of viral cyclin (v-Cyc), viral Bcl-2 (v-Bcl-2), viral macrophage inflammatory protein-I (v-MIP-I), viral interferon regulatory factor-1(v-IRF-1), and viral tegument protein (TP) was examined by RT-PCR Southern blot. Viral protein expression within the cells was examined by indirect immunofluorescence using 5 different HHV-8 positive antisera from 4 renal transplant recipients and 1 patient with classic KS. RESULTS: Gardella gel analysis revealed that HC induced an accumulation of the linear replicative genomic form of the virus in a time-dependent fashion. Southern blot analysis of the RT-PCR products revealed that HC induced increased expression of v-IRF-1, v-Bcl-2, and TP mRNA, with little discernible effect on v-Cyc, and v-MIP-I. Immunofluorescence revealed that HC induced increased numbers of cells expressing lytic antigens. CONCLUSIONS: These data indicate that hydrocortisone acts directly on BCBL-1 cells to activate the lytic cycle of HHV-8 and provide further support for the hypothesis that HHV-8 is activated in corticosteroid-treated immunocompromised patients.

Serologic and molecular evidence of human herpesvirus 8 activation in renal transplant recipients.

This study was designed to determine whether there is serologic or molecular evidence of human herpesvirus 8 (HHV-8) activation in renal transplant patients, an immunocompromised population at risk for development of Kaposi's sarcoma. Indirect immunofluorescence for detection of HHV-8 serum antibody and Southern blot polymerase chain reaction (PCR) for detection of viral DNA in whole blood were used. Seroprevalence and geometric mean titer (GMT) were significantly increased in the transplant group compared with healthy adults and were comparable to those in human immunodeficiency virus (HIV)-positive adults (transplant patients, 50% [GMT 1:210]; healthy adults, 7% [GMT 1:44]; HIV-positive patients, 73% [GMT 1:172]). Viral DNA was present in the blood of some renal transplant patients (3/33 PCR-positive) but in none of 20 healthy adults. Thus, there is both serologic and molecular evidence of HHV-8 activation in the renal transplant population compared with healthy adults (P<.01). The serologic results approximate those obtained for HIV-positive adults.

Pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with Epstein-Barr virus infection.

We report the clinicopathologic characteristics of pulmonary lymphomatoid granulomatosis (LYG) in 11 patients (identified from a series of 330 consecutive patients who underwent autopsy between 1984 and 1995 at the University of Texas Medical Branch at Galveston, Texas) with a diagnosis of acquired immunodeficiency syndrome (AIDS). We used immunohistochemical stains, RNA in situ hybridization (ISH), and gene rearrangement studies to identify the immunophenotype and the presence or absence of Epstein-Barr virus (EBV) infection. All of the patients were men ranging in age from 27 to 65 years (mean age, 38.6 yr). Autopsy lungs of 21 age-matched controls were examined for EBV using ISH; these included 9 patients with AIDS who did not have pulmonary lesions and 12 HIV-negative individuals who died accidentally (mean age, 38.6 yr). All of the 11 pulmonary lesions showed the gross and microscopic characteristics of LYG, with zonal necrosis and prominent angioinvasion. The tumor nodules consisted of a mixture of atypical large lymphocytes, with vesicular nuclei and prominent nucleoli and with a background of small and intermediate-size lymphocytes, histiocytes, and plasma cells. The large lymphocytes were CD20 positive, consistent with a B-cell phenotype. Ten of the 11 cases demonstrated EBV1-encoded RNA and CD20 positivity in the large, atypical lymphocytes by double labeling. One patient showed EBV positivity in CD20-negative, CD45RO-positive large cells, but these cells were CD3 negative and showed a monoclonal heavy chain gene rearrangement by polymerase chain reaction, indicating that these were of B-cell origin. Aberrant CD43 coexpression was identified in four cases. EBV latent membrane protein was demonstrated in 9 of 11 cases by immunohistochemical stains. The lungs of all of the 21 control patients were negative for EBV by ISH. We conclude that, in our series, AIDS-associated LYG is a B-cell neoplasm and that it has a strong association with EBV infection.

Detection of human herpesvirus-8 DNA in Kaposi's sarcomas from iatrogenically immunosuppressed patients.

BACKGROUND: Kaposi's sarcoma (KS) accounts for more than 5% of malignancies in immunosuppressed organ transplant patients (OKS). A new herpesvirus (HHV-8) was identified with high prevalence in biopsy specimens of AIDS-KS, endemic KS, and classic KS and in OKS. KS has also been associated with other underlying diseases in patients treated with corticosteroids, but this subset of KS has been reported to contain HHV-8 in only a few case reports. OBJECTIVE: In this larger study, we determined the prevalence of HHV-8 in seven patients of Jewish origin in whom KS developed during immunosuppressive therapy for different primary diseases (ISKS). METHODS: The study included HHV-8 DNA detection by polymerase chain reaction (PCR) coupled with Southern blot and sequence analysis as well as by in situ hybridization. RESULTS: HHV-8 sequences were detected by PCR with confirmation by Southern blot and sequence analysis in 100% of the ISKS samples. Direct sequencing revealed several previously unknown base changes within the 208 bp region from open reading frame 26 (ORF26[208]) of HHV-8 in ISKS. CONCLUSION: Ours is the largest known study describing the presence of HHV-8 in iatrogenic KS from immunosuppressed nontransplant patients and provides data of previously unknown sequence variations within the ORF26 of HHV-8 DNA.