Human epidermal Langerhans cells express beta 1 integrins that mediate their adhesion to laminin and fibronectin. Members of the beta 1 or very late antigen (VLA) integrin family represent the predominant class of integrin extracellular matrix receptors. Adhesion assays were developed for the identification of the beta 1 integrins involved in the adhesive interactions between Langerhans cells (which mainly express alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1) and extracellular matrix proteins. For this purpose, binding assays were performed on fibronectin-, laminin-, collagen type IV-, and collagen type I-coated plates. 59% +/- 21% of Langerhans cells (LC) specifically attached to fibronectin. Using as inhibitory probes monoclonal antibodies against the beta 1, alpha 5, and alpha 3 chains and the synthetic peptide GRGDSP resulted in a decrease of 43%, 41%, 15%, and 42% respectively of LC binding to fibronectin. 76% +/- 20% of LC specifically adhered to laminin. Anti-alpha 6 monoclonal antibody potently inhibited this adhesion, which dropped to 36%, whereas the synthetic peptide GRGDSP was ineffective. A low number of LC adhered to type I and type IV collagen (13-15%). These results indicate that alpha 5 beta 1 and alpha 6 beta 1 were the major beta 1 integrins involved in LC adhesion to fibronectin and laminin. Ultrastructural cell morphology of adherent cells was examined and showed that LC were largely spread on laminin and became tightly bound to the substrate on a large portion of membrane. On fibronectin surface, the contact between LC and substrate was smaller, thus cells could conserve their general round aspect. Moreover, LC binding to fibronectin and laminin induced a significative decrease of the Birbeck granule number. The finding that LC attach to LM and FN in vitro suggests they exist similarly in vivo. By mediating a passage through basement membrane and migration throughout the fibronectin network of the dermis, alpha 5 beta 1 and alpha 6 beta 1 could contribute to the ability of LC to migrate into and out of the epidermis.


Release of soluble Fc gamma RII/CD32 molecules by human Langerhans cells: a subtle balance between shedding and secretion? Freshly isolated human Langerhans cells (LC) express two forms of Fc gamma RII: a membrane-associated form detected by monoclonal antibody (MoAb) anti-CD32, which recognize an extracytoplasmic epitope of the molecule, and a soluble secreted form, whose existence is suggested by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments. Indeed, RT-PCR performed on total LC RNA reveals the presence of two Fc gamma RIIA mRNA, one encoding the FC gamma RIIA with a transmembrane region (membranous form) and the other without this region (soluble form). Densitometry studies performed on the two PCR products reveal that the ratio between the membranous form and the soluble secreted form is about 1.5. LC maintained in culture for 24-48 h lose the major part of their membrane Fc gamma RII expression (shown by flow cytometry) and release soluble Fc gamma RII molecules (revealed by dot-blot assay), but maintain the same ratio of the two Fc gamma RIIA mRNA. The disappearance of the membrane-associated Fc gamma RII may be explained either by modification of its recycling pathway or by proteolytic cleavage of the receptor at the cell surface. Thus, soluble Fc gamma RII molecules generated during LC culture may result from proteolytic cleavage of the cell-surface receptor and/or secretion of a soluble form derived from the translation of an alternate spliced mRNA. Interestingly, addition of TNF-alpha (10 ng/ml) to the culture medium i) maintains the expression of the membranous form, which can be detected on the LC surface at the same level as on freshly isolated LC, and ii) reverses the ratio (to 0.6) of the two Fc gamma RII mRNA, the mRNA encoding the soluble form becoming predominant. Thus, TNF-alpha seems to modify the expression of the Fc gamma RII at the mRNA level, favoring the secretion of soluble Fc gamma RII molecules, and changes the fate of the membranous Fc gamma RII.


Membrane ecto-ATPase on epidermal Langerhans cells. Epidermal Langerhans cells (LC) possess a plasma membrane Mg(++)- or Ca(++)-dependent ecto-ATPase (mATPase), the function of which is not known. On the other hand, extracellular ATP (ATPo) has been shown to cause a rapid increase in cell-membrane permeability for selected cells, thus inducing the entry of exogenous substances, leading eventually to cell death. ATPo has also been shown to be a mediator of T cell-mediated cytotoxicity. We have observed human LC to be resistant to the permeabilizing effects of ATPo when compared to cultured human keratinocytes and J774 macrophages. In addition, our preliminary evidence in vitro suggests that mATPase on LC may provide protection against the lytic effects of ATPo. Finally, we propose that this latter mechanism may also operate in vivo.


Characteristics of antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice. Exposure of mice to ultraviolet (UV)-B (280-320 nm) radiation alters their immune response to contact-sensitizing haptens applied to UV-irradiated skin. Under these conditions, the contact-hypersensitivity response is reduced, and hapten-specific suppressor T lymphocytes can be found in the spleen. Considerable evidence suggests that the epidermal Langerhans cell is one of the principal targets of this effect of UV irradiation that leads, ultimately, to suppressor cell formation. We are using a combination of cell-surface markers and light and electron microscopy to determine whether differences can be detected in the antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.


Alpha IIB beta 3 integrin dissociation induced by EDTA results in formation of Birbeck granule-like structures in human blood platelets. EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces morphologic changes in the platelet surface-connected canalicular system (SCCS), in particular the collapse of some portions of the canaliculi. These collapsed elements are made up of two parallel limiting membranes and a central irregular striated zone, a pentalaminar organization that clearly resembles that of Langerhans cell Birbeck granules (BG). Such BG-like structures are also seen between adjacent platelets in EDTA-treated platelet microaggregates. EDTA is known to induce an irreversible dissociation of the platelet membrane glycoprotein(GP)IIb-IIIa, the alpha IIb beta 3 platelet-specific intergrin, a calcium-dependent heterodimer that serves as an inducible receptor for fibrinogen and is essential for platelet aggregation. Hence, we looked for involvement of the GPIIb-IIIa in the formation of these BG-like modifications. We observed that these changes i) cannot be induced in type I Glanzmann's thrombasthenia, where the GPIIb-IIIa complexes are absent; ii) did not appear when human platelets were pre-incubated with MoAb anti-GPIIb-IIIa complex, which protected GPIIb-IIIa from EDTA-induced dissociation; iii) appeared only at alkaline pH and 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate the GPIIb-IIIa complexes; iv) are accompanied by the dissappearance on fluorescence flow cytometry analysis of the heterodimer specific epitopes; and v) do not appear in rat platelets at pH 7.4 where GPIIb-IIIa does not dissociate after EDTA treatment. Thus, the appearance of BG-like structures in the platelet SCCS is directly dependent on the EDTA-induced dissociation of the GPIIb-IIIa complexes. Furthermore, using gold-labeled MoAb concomitantly with the addition of EDTA, we observed that only GPIIb is present in the collapsed portions of the canaliculi. On Lowicryl thin sections essentially polyclonal antibodies to GPIIb labeled the central striated zone. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS and raise the question of the participation of a Langerhans cell integrin in the formation of Langerhans cell BG.


Fc epsilon RI mediates IgE binding to human epidermal Langerhans cells. In a recent series of experiments, we observed that epidermal Langerhans cells (LC) of healthy, non-atopic individuals have the capacity of specifically binding monomeric serum or myeloma IgE. IgE-binding to LC could neither be prevented by pre-incubation of the cryostat sections with monoclonal antibodies (MoAb) against either Fc epsilon RII/CD23 or Fc gamma RII/CD32 nor by the addition of excess amounts of lactose, but could be entirely abrogated by pre-incubation with the anti-Fc epsilon RI MoAb 15-1. A direct testing of the anti-Fc epsilon RI MoAb 15-1 and 19-1 on cryostat sections in an indirect immuno-double-labeling technique showed that, in contrast to eight different anti-Fc epsilon RII/CD23 MoAb, these MoAb react with the majority of CD1a-bearing epidermal cells. At an ultrastructural level, 15-1 immunogold-labeling in the epidermis was confined to the surface of cells exhibiting Birbeck granules. In further experiments, we were able to amplify by polymerase chain reaction (PCR) technology transcripts for the alpha, beta, and gamma chains of Fc epsilon RI from LC-enriched epidermal cells and dermal cells, but not from LC-depleted epidermal cells. Transcripts for the mast cell enzyme tryptase were exclusively found in dermal cell-derived RNA preparations, thus excluding a contamination of the LC-enriched epidermal cell preparations by dermal mast cells. Collectively, these data show that epidermal LC, but not other epidermal cells, express Fc epsilon RI molecules.


Development and function of dendritic cells in health and disease. The life history of dendritic cells (DC) is now established from their origins from bone marrow stem cells, their distribution through blood to the tissues, and their movement via afferent lymph to lymph nodes for the initiation of immune responses. Bone-marrow stem cells, and occasional stem cells in peripheral blood (about 1 per 10(5) mononuclear cells), can give rise both to DC and macrophages (MO). In addition to stem cells in blood, after short-term culture of mononuclear cells, three major morphologic types of DC can be separated (types I-III), which probably represent the maturational pathway of this cell type; type II cells resemble tissue DC such as Langerhans cells and type III have a veiled morphology similar to that seen in cells of afferent lymph and in the interdigitating cells of the paracortex of lymph nodes. Functionally, DC cultured from peripheral blood are able to acquire large antigens and process them like Langerhans cells of the skin. They can also present antigens to stimulate primary T-cell responses, a property associated with lymph node DC. In tissues, DC appear to act as outposts of the immune system, acquire antigens, and, particularly in primary responses, carry the antigens to lymph nodes where they initiate T-cell responses. In secondary responses, activation of memory T cells in the periphery and the acquisition of antigen/antibody complexes by follicular dendritic cells of the lymph node follicles, which stimulate B cell memory, may be more important pathways for immune activation. DC may play a role in the development of many immunologic diseases including cancer, autoimmunity, and acquired immunodeficiency syndrome (AIDS).


Cytokine expression by epidermal cell subpopulations. Epidermal cells (EC) are a rich source of cytokines that can regulate the function of cells in skin and in other tissues. To organize the array of data pertaining to cytokine expression by EC subpopulations, we have tabulated such data according to cell source, state of cell activation, and type of assay employed. This information forms a background for our own studies, in which reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show that Langerhans cells (LC) are the principal source of mRNA for interleukin 1 beta and macrophage inflammatory protein-1 alpha (MIP-1 alpha) among unstimulated mouse EC.


Differentiation of epidermal Langerhans cells in macrophage colony-stimulating-factor-deficient mice homozygous for the osteopetrosis (op) mutation. The osteopetrosis (op) mutation is within the gene for macrophage colony-stimulating factor (M-CSF). Homozygotes (op/op) lack M-CSF activity and show abnormalities in the differentiation of osteoclasts and other cells within the macrophage lineage. The effect of the op mutation on the development of Langerhans cells (LC) was determined in order to assess differentiation of such cells in vivo in the absence of M-CSF. (C57BL/6J X C3HeB/FeJ)F2-op/op and +/? Littermate control mice were raised from +/? breeders obtained from the Jackson Laboratory. The mice were killed with ether anesthesia at 4 weeks after birth and skin specimens were excised and examined by immunohistochemistry using anti-mouse pan macrophage monoclonal antibodies (MoAb), F4/80 and BM8; anti-mouse LC MoAb, NLDC-145, M1-8, and MIDC8; and anti-mouse Ia MoAb, M5/114. In epidermal sheets, numbers of LC were counted. Histochemical staining of adenosine diphosphatase (ADPase) localization was also performed as a marker of LC. Epidermal LC from op/op mice showed reactivity with all these MoAb. Numbers of LC were slightly reduced, but the reduction was not significant statistically. The presence of Birbeck granules in LC of op/op mice was confirmed by electron microscopy but the cytoplasmic projection of LC was not prominent. From these results, it appears obvious that the development and differentiation of LC do not require M-CSF.


Stimulation of Langerhans cell migration by tumor necrosis factor alpha (TNF-alpha). Following topical exposure of mice to skin-sensitizing chemicals, Langerhans cells (LC), many of which bear antigen, are stimulated to migrate via the afferent lymphatics to draining lymph nodes. Consistent with the acquisition of potent immunostimulatory activity, LC while in transit to lymph nodes, are subject to a functional and phenotypic maturation thought to be mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) and possibly other epidermal cytokines. An interesting question is the nature of the stimulus that initiates the migration of LC from the epidermis. We have examined the influence of intradermal tumor necrosis factor alpha (TNF-alpha), another epidermal cytokine, on the accumulation of dendritic cells (DC) in draining lymph nodes. Murine, but not human, recombinant TNF-alpha caused a rapid and concentration-dependent increase in the frequency of DC in draining nodes. The conclusion drawn is that local production of TNF-alpha provides one signal for LC migration during cutaneous immune and inflammatory responses.


Migration of Langerhans cells from carcinogen-treated sheep skin. To define the mechanism(s) of carcinogen depletion of Langerhans cells (LC) from skin, the migration of LC from the skin to the regional lymph node was examined in carcinogen-treated, antigen-treated, and control sheep. This was assessed by cannulation of afferent lymphatic vessels that drain the treated areas of skin or the efferent lymphatic draining the regional lymph node. Cells draining from test or control skin were continuously collected and enumerated by indirect immunofluorescence and flow cytometry using specific anti-CD1 monoclonal antibodies. There was a marked increase in the rate of LC migration in the 8 h following the application of the contact sensitizing antigen trinitrochlorobenzene (TNCB). The chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) triggered a tenfold-greater migration of LC compared with TNCB--with the peak response at 5 d. After DMBA treatment LC were also detected in the efferent lymph of the regional lymph node. It is concluded that the depletion of LC from carcinogen-treated skin is due to the increased LC migration and not carcinogen-induced cell death.


Migration of Langerhans cells into the epidermis of human skin grafted into nude mice. In a previous study, it was demonstrated that human Langerhans cells (LC) are preserved in human skin grafted onto a nude mouse. Moreover, although it was observed that mouse LC of the host invade skin grafts from allogeneic mouse or rat, they do not penetrate in human skin grafts. In most of the human skin equivalent systems produced in vitro, LC appear to be lost. The present study was designed to investigate whether the mouse LC will repopulate a human skin equivalent. For this purpose, two different systems of skin equivalent have been grafted into the nude mouse. They were composed of human keratinocytes deposited on dead human dermis, or on lattice composed of human fibroblasts embedded in type I collagen. At different times after grafting, the presence of LC in the transplants was assayed either by indirect immunofluorescence or by electron microscopy. Indirect immunofluorescence was performed on frozen sections or on epidermal sheets with anti-Ia, anti-HLA-DR, or OKT6 antibodies. It was observed that, at 2 months after grafting, Ia(+) HLA-DR(-) OKT6(-) cells are present in grafted human epidermis. Moreover, LC with typical Birbeck granules are also detected by electron microscopy. It could be concluded, from this study, that mouse LC can repopulate human epidermis devoid of human LC.


Cytokine-stimulated human dermal microvascular endothelial cells produce interleukin 6--inhibition by hydrocortisone, dexamethasone, and calcitriol. The effects of lipopolysaccharide (LPS), recombinant human tumor necrosis factor-alpha (TNF), recombinant human interleukin 1-beta (IL-1 beta), and interferon-gamma (IFN-gamma) on IL-6 production were determined by enzyme-linked immunosorbent assay (ELISA) and by Northern blot analysis in cultured human dermal microvascular endothelial cells (HDMEC). Unstimulated HDMEC did not produce significant amounts of IL-6, whereas lipopolysaccharide (LPS), TNF, and IL-1 beta were potent inducers of HDMEC-derived IL-6 production. Treatment with IFN-gamma had no effect. IL-1 beta stimulation resulted in pronounced IL-6 production after 4 h, followed by complete downregulation at the transcriptional level after 24 h. In contrast, LPS and TNF induced prolonged stimulation of IL-6 production by HDMEC as IL-6 mRNA transcripts were still detected after 24 h treatment and IL-6 protein was markedly increased at this timepoint. The effects of hydrocortisone, dexamethasone, calcitriol, acitretin, and cyclosporin A on TNF- or IL-1 beta-induced IL-6 production by HDMEC were determined by ELISA. Both hydrocortisone and dexamethasone dose-dependently inhibited the cytokine-induced IL-6 production, whereas the inhibition by calcitriol was less pronounced. In contrast, acitretin and cyclosporine A had no influence on cytokine-induced HDMEC IL-6 production. These results disclose dermal endothelial cells as a major source for the pro-inflammatory cytokine IL-6, involved in the regulation of inflammatory skin processes. As IL-6 seems to play a key role in the pathogenesis of psoriasis, the beneficial effects of corticosteroids and calcitriol in this disease may partly be explained by their ability to inhibit HDMEC-derived IL-6 production.


Mapping of the trichohyalin gene: co-localization with the profilaggrin, involucrin, and loricrin genes. The chromosomal location of the gene encoding the human hair follicle protein trichohyalin has been determined by in situ hybridization. The human gene has been localized to the region 1q21.1-1q23 (probably 1q21.3) using a sheep trichohyalin cDNA probe. The genes encoding three other epithelial proteins, namely, profilaggrin, involucrin, and loricrin, are also located in the same region of chromosome 1, which, together with their similar gene and protein structures, suggests that the four proteins form a novel superfamily of epithelial structural proteins.


Anchoring fibrils, collagen VII, and neutral metalloproteases in recessive dystrophic epidermolysis bullosa inversa. Structure of the anchoring fibrils, expression of collagen VII, and gelatinolytic activity in skin fibroblasts were assessed in six patients with epidermolysis bullosa dystrophica (EBD) inversa and in control groups consisting of probands with other EBD subtypes and healthy individuals. All six patients with EBD inversa, as well as the patients with generalized non-mutilating and localized EBD, showed positive staining with antibodies to collagen VII, the major anchoring fibril protein. Four patients with severe generalized mutilating EBD exhibited negative staining. Ultrastructurally, normal anchoring fibrils were demonstrable in uninvolved skin of patients with localized, inversa, and generalized non-mutilating subtypes. At the same time, a high degree of variability was observed in the amount and quality of anchoring fibrils in the various stages of lesional skin, including co-existence of normal and partially degraded anchoring fibrils. Of all 12 patients only one localized and two inversa cases showed an increased gelatinolytic activity in vitro. However, the high activity was associated with neither the severity of the disease nor the inversa subtype. In addition, intact collagen VII could be extracted from the dermis of one inversa patient. The present data show no correlation between increased in vitro gelatinolytic activity and abnormalities of the anchoring fibrils or collagen VII in skin of patients with recessive EBD, and therefore suggest molecular heterogeneity of the causative pathogenetic mechanisms.


Characterization of biologic properties of wound fluid collected during early stages of wound healing. The clinical effects of occlusive dressings on wound healing are well documented. However, the underlying biologic mechanisms associated with moist healing are not well understood. Experimental studies and clinical experience have shown enhanced eschar and clot removal, re-epithelialization, and collagen synthesis under occlusion, suggesting the possibility of elevated activities of proteinases and other effectors, e.g., growth factors, in the moist wound environment. To gain an insight into the biology of early wounds under occlusion, we have carried out biologic and biochemical analyses on fluids from occluded full- and partial-thickness wounds. Metalloproteinase activities were detected in the wound fluid samples. When applied to cultured dermal fibroblasts, mitogenic activity was observed with fluids from full-thickness wounds. Wound fluid-stimulated accumulation of urokinase-type plasminogen activator by fibroblasts was also observed in a time-dependent manner. Stimulation of metalloproteinase accumulation by fibroblasts was also observed. We have further demonstrated the presence of platelet-derived growth factor-like and basic fibroblast growth factor-like factors in wound fluid by antibody neutralization of their biologic activities. Proteinase presence and proteinase stimulatory activity of wound fluid retained in the occluded wound may contribute to an enhanced proteolytic environment in these wounds in comparison to non-occluded "dry" wounds. The presence of growth factors and the potential abilities of proteinases to activate latent growth factors and generate chemotactic peptides through connective tissue breakdown may also contribute to the enhanced healing of occluded wounds.


A novel monoclonal antibody to a distinct subset of cutaneous dendritic cells. A monoclonal antibody was generated by immunizing rats with Langerhans cell (LC)-enriched epidermal cells obtained from BALB/c mouse earskin after epicutaneous application of the contact sensitizer 2,4-dinitrofluorobenzene (DNFB). The antibody 4F7 detects in normal mouse skin, few dermal cells showing the morphologic, phenotypic, and functional properties of accessory dendritic cells, but lacking Birbeck granules. The capacity to stimulate allogenic T cells in the mixed leucocyte reaction resembles that of freshly isolated LCs. After DNFB application, an increased number of 4F7+ dendritic cells are found in the dermis and, in addition, some labeled dendritic cells occur in the epidermis. Some of the latter cells exhibit cytoplasmic Birbeck granules. Remarkably, there is no increase of the 4F7+ cells in the regional lymph nodes after DNFB treatment. These data suggest that the 4F7 antibody labels distinct dendritic cells of the mouse skin that are involved in the mediation of contact sensitization and probably represent immature LCs.


Protein kinase C in normal human epidermal keratinocytes during proliferation and calcium-induced differentiation. Recent evidence has implicated protein kinase C (PKC) in the etiology of hyperproliferative diseases such as psoriasis and non-melanoma skin cancer. In this study, PKC activity, immunoreactive protein, and phorbol ester-binding kinetics were examined in primary cultures of normal human epidermal keratinocytes (NHEK) in order to elucidate the relationship between PKC and NHEK proliferation and differentiation. NHEK were maintained in a proliferative phase in serum-free low-calcium (0.15 mM) medium, and then were exposed to high calcium (1.6 mM) in order to stimulate growth arrest and differentiation. Staurosporine was inhibitory to Ca(++)-induced differentiation. Scatchard analysis of phorbol binding indicated that exposure to high calcium for 24 h increased the number of binding sites (Bmax) by fivefold. In correlation with the ligand-binding results, PKC activity was extremely low in proliferating (low-calcium) NHEK compared to differentiating cells (high calcium). When assayed after 24, 48, and 72 h, high calcium induced tenfold or greater increases in Ca++/phospholipid-dependent phosphotransferase activity. Immunoblot analysis of NHEK PKC using antibodies directed against the hinge region of PKC alpha/beta also indicated that exposure to high calcium resulted in higher levels of immunoreactive protein. Therefore, PKC in NHEK appears to be upregulated under conditions of Ca(++)-induced growth arrest and differentiation. In addition, NHEK and other human skin cell particulate fractions contain a protein of approximately 116 kDa that is highly immunoreactive to an antibody to PKC alpha/beta, which coelutes from DEAE-sephacel under the same buffer conditions as the 80-kDa PKC.


Scanning electron microscopy of the epidermal lamina densa in normal human skin. The lamina densa of normal human epidermis was exposed by treatment with 1 M sodium chloride and was examined by high-power scanning electron microscopy before and after trypsinization. Localization of type IV collagen in the lamina densa was also studied by transmission and scanning immunoelectron microscopy. Before trypsinization, the surface of the lamina densa consisted of microridges and microvalleys. The microridges varied in height and were connected with each other. They were arranged in a concentric fashion around the tips of the dermal microprojections. At a higher magnification, the surface of the lamina densa was composed of densely packed cobblestone-like structures approximately 7-15 nm in size, between which were interspaces 4-11 nm wide. These structures expressed type IV collagen. After trypsinization, the lamina densa was found to be composed of microfilaments approximately 10 nm thick showing beaded appearances. These microfilaments exhibited the same cobblestone-like structures as the lamina densa surface. Observation of the torn lamina densa demonstrated anchoring fibrils and oxytalan fibers that were attached to the lamina densa itself. Another kind of filament about 7 nm thick linked the anchoring fibrils and the oxytalan fibers. Beneath the lamina densa was a network of fibers about 40-50 nm thick, which was composed of collagen fibers and possibly also elaunin fibers. In conclusion, this study revealed the detailed surface ultrastructure of the epidermal lamina densa and its underlying filamentous elements.


Fibronectin gene transcription is enhanced in abnormal wound healing. Abnormal wound healing in susceptible individuals can result in the formation of keloids that have an elevated content of extracellular matrix material compared to normal scars. Keloid-derived fibroblasts exhibit as much as a four-fold increase in the rate of fibronectin biosynthesis compared to fibroblasts from normal dermis and normal scars. Altered biosynthesis is due to an increase in the steady-state level of fibronectin mRNA, and in this investigation we have identified the level of fibronectin gene expression that is responsible for this increase. The rate of fibronectin gene transcription was found to be increased as much as threefold in keloid fibroblasts when compared to normal fibroblasts. Other possible changes that could account for the elevated level of fibronectin mRNA in keloids, such as increased copy number of the fibronectin gene or decreased turnover of fibronectin mRNA were also examined. The possibility of altered gene dosage was eliminated because chromosome content, G-banding patterns, and fibronectin gene content of keloid fibroblasts were all found to be normal. Analysis of fibronectin mRNA degradation revealed a half-life of approximately 13 h, and the residual fibronectin mRNA was observed to remain full length during this time period in both keloid and normal fibroblasts. Thus, altered degradation of fibronectin mRNA is unlikely to contribute to overproduction of fibronectin in keloids. Increased translational competence of fibronectin mRNA in keloids was also eliminated as a contributing factor because fibronectin mRNA remaining after one half-life were equally available for translation in both cell types. Although stimulation of transcription may not entirely account for the increase in fibronectin biosynthesis in keloids, this mechanism is best able to account for the majority of the change.


Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts. Immigration of Langerhans cell precursors from the peripheral blood to the skin was studied in human grafts placed on severe combined immunodeficient (SCID) mice. Monocyte fractions of human blood were injected intraperitoneally to SCID bearing either reconstituted (Langerhans cell free) epidermal sheets (E) or living skin equivalents (E/D) consisting of both epidermis and dermis. A range of immunocytochemical and ultrastructural markers was employed to monitor the colonization of the grafts, i.e., CD1a/c, Birbeck granules. In situ hybridization with probes against Alu sequences of human DNA were employed together with immunostaining for MHC class I mouse and human antigens to document graft survival. Although unequivocal LC were detected within E grafts, including both human (CD1a positive) and murine (NLDC-145 positive), no migration was achieved in the E/D situations.


Inhibition of cutaneous contact hypersensitivity in the mouse with systemic or topical spiperone: topical application of spiperone produces local immunosuppression without inducing systemic neuroleptic effects. We tested the ability of the neuroleptic agent spiperone (8-[3-(p-fluorobenzoyl)propyl]-1-phenyl-1,3,8-triazaspiro-[4.5] decan-4- one) to influence the tissue swelling and leukocyte infiltration associated with T-cell--dependent immune responses, i.e., contact hypersensitivity reactions, in mice. Contact hypersensitivity reactions were elicited by applying the haptens oxazolone or dinitrofluorobenzene topically to one or both ears 5-8 d after epicutaneous sensitization. When spiperone was given subcutaneously at a dose of 30 or 150 mg/kg, 1 h after challenge with oxazolone, cutaneous contact hypersensitivity to this hapten was significantly diminished. When applied topically in concentrations as low as 0.08% (w/w), preparations of spiperone significantly suppressed both the tissue swelling and the leukocyte infiltration associated with the elicitation phase of contact hypersensitivity. Topical treatment with spiperone also suppressed the sensitization phase of contact sensitivity. However, mice treated topically with spiperone, unlike those treated systemically, exhibited no drowsiness or other evidence of central nervous system effects. Spiperone expresses both serotonin and dopamine receptor antagonist activity. However, unlike spiperone, the chemically unrelated serotonin antagonists, trazadone and mianserin, and the dopamine receptor antagonist, haloperidol, were not effective in suppressing contact hypersensitivity. Our results indicate that spiperone can have immunosuppressive effects on contact hypersensitivity reactions in the mouse, even when applied topically in doses that lack neuroleptic effects, and that the mechanism of action of spiperone on the immune response may be independent of its serotonin or dopamine receptor blocking properties.


Persistent immune tolerance to nickel and chromium by oral administration prior to cutaneous sensitization. Oral administration of allergens, foreign proteins, or cell-bound antigens may induce systemic suppression of subsequent humoral and cell-mediated immune responses ("oral tolerance"). The induction of specific immune tolerance provides a potential strategy for treatment of T-cell-dependent immune diseases. Therefore, in depth studies into preconditions for optimal and persistent tolerance induction are mandatory. Here we report on such studies in a guinea pig model using the non-cross-reactive contact allergens nickel and chromium. Feeding per os of nickel sulfate or potassium dichromate did not trigger systemic TDTH-effector functions. Instead, short feeding periods led to a dose-dependent, and metal-specific, suppression of subsequently induced allergic contact hypersensitivity. Administration of the allergens onto the oral mucosa was most effective in the induction of immune tolerance. When first sensitizing attempts were delayed until 1 year after feeding, the degree of unresponsiveness was reduced. In contrast, with cutaneous contacts starting shortly after the feeding period, tolerance was fully stable and undiminished for at least 2 years. Thus, in orally treated guinea pigs cutaneous contacts provide boosting tolerogenic signals, supporting the view that oral tolerance does not result from clonal deletion but from active antigen-specific immunosuppression. Indeed, unresponsiveness to cutaneous immunization could be transferred by lymphoid cells from fed guinea pigs in a metal-specific way.


Composition of free long-chain (sphingoid) bases in stratum corneum of normal and pathologic human skin conditions. The composition of molecular species of free long-chain bases (FLCB) isolated from stratum corneum of various human skin conditions was analyzed by the high-performance liquid chromatographic and fast atom bombardment mass spectrometric methods. FLCB with carbon length ranging from 16 to 20 comprised about 0.3% of total lipids in stratum corneum of normal and pathologic skin conditions. Major FLCB included sphinganines and sphingenines with 18 to 20 carbons and some phytosphingosines such as t17:1, t18:1, t18:0, t20:1, and t20:0. Compared with stratum corneum of normal lower legs, molar percentages of FLCB having 18 carbons and those with 20 carbons were slightly higher and lower, respectively, in normal plantar epidermis, showing site-related differences in normal skin. Psoriatic scales and hyperkeratotic stratum corneum from clavus and plantar keratoderma contained increased levels of FLCB with 18 carbons and decreased levels of FLCB with 20 carbons. These findings may reflect abnormal keratinization in hyperkeratotic skin conditions.


Induction of the 72-kD heat shock protein in xeroderma pigmentosum complementation group A fibroblasts. In mammalian cells, 72-kD heat shock protein (HSP72) is the major stress-inducible protein that is thought to play a protective role against the various environmental stresses. In order to know the induction mechanism of HSP72, we examined the HSP72 in DNA repair-deficient xeroderma pigmentosum group A fibroblasts (XP2OSSV) and normal fibroblasts (WI38VA13) by the indirect immunofluorescence method using a monoclonal antibody specific for the inducible 72-kD protein. Heat-shock treatment of the same survival fraction (5% survival) induced HSP72 in xeroderma pigmentosum (XP) and normal cells. However, as compared with XP cells, normal cells showed the induction of HSP72 more rapidly and strongly. When XP and normal cells were irradiated with UVC at the same survival dose (10% survival), apparent induction of HSP72 was observed in both cell lines. In the case of UVC irradiation at the same dose (1.0 J/m2), though XP cells showed the induction of HSP72, HSP72 was not induced in normal cells. In both cell lines, heat-shock treatment caused more rapid induction of HSP72 than UV irradiation. These results suggest that the induction mechanism of HSP72 might be different between heat-shock treatment and UV irradiation. In addition, in the case of UV irradiation, the extent of DNA damage after DNA repair or the cell death might be involved in the induction of HSP72.


Proteoglycans synthesized by adult human epidermis in whole skin organ culture. Adult human epidermis was cultured in whole skin organ culture under serum-free conditions in the presence of 35SO4. Proteoglycans (PG) comprised about 25% of the total (35SO4)-labeled material produced by epidermis. The rest of the incorporated activity displayed solubility characteristics typical of lipids. The molecular mass and the composition of the 35SO4-labeled epidermal PG and glycosaminoglycans (GAG) were studied using gel filtrations and agarose gel electrophoresis. The 35SO4-label of the epidermal PG was located in heparan sulfate (HS, approximately 75%) and chondroitin/dermatan sulfate (CS/DS, 25%), but not in keratan sulfate as determined by nitrous acid, chondroitinase AC II, chondroitinase ABC, and keratanase digestions, respectively. The molecular mass of the GAG chains was 10-40 kDa. The 35SO4-labeled PG were distributed between 60 and 600 kDa in agarose gel electrophoresis, with the highest activity at 350 kDa. Smaller activity peaks occurred at 150 and 60 kDa. Digestion of the PG with heparitinase removed most of the activity at 350 and 150 kDa, whereas chondroitinase ABC removed that at 60 kDa. A small amount of activity migrating between 600 and 1000 kDa was not affected by any of the GAG-degrading enzymes. Pulse chase experiments showed that the epidermal PG had an average half life of 24 h. The results thus demonstrate that human epidermis produces at least three different, rapidly metabolized PG. The PGs from 150 to 350 kDa contained heparan sulfate chains, whereas those at 60 kDa were chondroitin/dermatan sulfate PG.


Presence of amelanotic melanocytes within the outer root sheath in senile white hair. In order to determine whether amelanotic melanocytes are present in senile white hair, we attempted to detect tyrosinase mRNA and its protein using in situ hybridization and immunohistochemical staining. Specifically stained cells containing tyrosinase mRNA or its protein were detected by each technique in 7/23 (30.4%) or 4/14 (28.6%) senile white hairs, respectively. These cells were located in the outer root sheath between the hair-bulb and the infundibulum. Silver staining was used to determine whether melanin was present within the outer root sheath of the senile white hair. Because none of the 21 white hairs tested showed specific staining within the outer root sheath, the absence of melanin was confirmed. Thus our results suggest the presence of amelanotic melanocytes within the outer root sheath of senile white hair.


Differential expression of calgranulin A and B in various epithelial cell lines and reconstructed epidermis. The monoclonal antibody F12, raised against epidermal cells from a psoriatic lesion, decorated antigens highly expressed in psoriatic epidermis and in cultured normal human keratinocytes. In normal human skin, F12 reacted only with follicular keratinocytes. Characterization of the immunoprecipitated antigens by two-dimensional gel electrophoresis revealed their identity with calgranulin A and B. A semiquantitative study with various established epithelial cell lines demonstrated that the expression of calgranulin A and B in hyperproliferative keratinocytes correlates with their potential to undergo terminal differentiation. In epidermis reconstructed in vitro, the antigen expression was stimulated by retinoids and suppressed under vitamin A starvation.


Quantitative determination of EGF-R during epidermal wound healing. Little is known about the intrinsic regulation of growth factors of cytokines during the normal epidermal wound-healing processes in skin. A simplified model of wounding (tape stripping to remove the stratum corneum) was used to study the role of epidermal growth factor receptors (EGF-R) in this process. Although the dynamics of EGF-R in epidermal wound healing have not been determined, the immunoreactive EGF-R that are present presumably play an active role. Prior studies show that 1) EGF-R are present in increased numbers in proliferative skin diseases; 2) a hypertrophic epidermis, closely resembling normal wound healing, is induced in mouse skin by EGF injections; and 3) exogenous topical EGF potentiates wound healing. The number of immunoreactive receptors as measured by an enzyme-linked immunosorbent assay (ELISA) and histologic methods increased prior to an increase in epidermal thickness, total protein, and DNA content. This early increase in the levels of EGF-R was followed by a sharp decline in EGF-R and subsequent decline in epidermal thickness (hypertrophy), total protein, and DNA levels. Alterations in the temporal sequence in these parameters indicate that the EGF-R-mediated signaling systems play an active role in epidermal wound repair.


Keloid fibroblasts exhibit an altered response to TGF-beta. Treatment with transforming growth factor beta 1 (TGF-beta 1) results in stimulation of total protein synthesis in normal dermal fibroblasts but not in keloid fibroblasts, suggesting that the TGF-beta regulatory program is altered in keloid fibroblasts. However, both keloid and normal fibroblasts treated with TGF-beta 1 exhibit accelerated fibronectin biosynthesis, indicating that keloid cells can respond to TGF-beta 1. In the absence of serum, the TGF-beta 1-induced increase in fibronectin biosynthesis occurs more rapidly in keloid fibroblasts, also suggesting modification of this regulatory pathway. The TGF-beta 1-mediated increase in keloid fibronectin production is independent of the steroid regulatory pathway for fibronectin, which accelerates synthesis by means of a post-transcriptional mechanism. Thus, TGF-beta 1 stimulation of fibronectin production in keloid cells is likely to involve a transcriptional mechanism and keloid overproduction of extracellular matrix components may be due to an inherent modification of the TGF-beta regulatory program.


Keratinocyte migration is partially supported by the cell-binding domain of fibronectin and is RGDS-dependent. Fibronectin (FN) plays a key role in cell attachment, embryonic development, and wound healing. In this respect, it is known that FN promotes keratinocyte migration. The aim of this study was to examine specific FN domains (120-kD cell-binding fragment, 45-kD collagen fragment, and 40-kD heparin fragment) and a biologically active peptide within the molecule (RGDS) for their ability to influence human keratinocyte (HK) locomotion. HKs were plated on gold salts coated with different substrates (type IV collagen, FN with or without the RGDS peptide, and the three FN fragments). After 20 h, locomotion tracks were quantified by computer-assisted image analysis that determines the area of each microscopic field occupied by migration tracks, a so-called migration index (MI). MIs on type IV collagen and FN were 39.14 +/- 2.8% and 30 +/- 0.4%, respectively. The maximal MIs on the collagen-binding domain and heparin-binding domain of FN were similar to our negative controls (plastic and albumin): 3 +/- 1%. In contrast, the maximal MI on the cell-binding fragment of FN was 18.45 +/- 2.1%. The effect of the cell-binding domain on keratinocyte motility was found to be dose dependent. Moreover, we could specifically inhibit the FN-driven locomotion using the RGDS sequence contained in the cell-binding fragment. We did not observe a synergistic effect (i.e., a higher MI) when we added the three fragments in a same dish. These results suggest i) that the cell-binding fragment of FN partially supports HK locomotion, ii) that other untested FN domain(s) should act in synergy with the cell-binding fragment to promote keratinocyte locomotion, or alternatively iii) that the FN function of promoting cell migration resides within the FN cell-binding domain, but the proper presentation of this domain to the cell requires an intact, native FN molecule, and iv) that the RGDS sequence is essential for HK movement.


Effects of immunomodulatory cytokines on the presentation of tumor-associated antigens by epidermal Langerhans cells. The recognition and presentation of tumor-associated antigens by cutaneous antigen-presenting cells (APC) may play an important role in the establishment of effective defense mechanisms against newly emerging tumors in the skin. Recent data demonstrate the ability of I-A+ epidermal cells (Langerhans cells) to present tumor-associated antigens for the induction of protective tumor immunity and elicitation of delayed-type hypersensitivity against the murine spindle cell tumor, S1509a. Furthermore, the local cytokine microenvironment in the vicinity of a cutaneous neoplasm may regulate the ability of resident epidermal APC to initiate and/or to elicit protective immunity against incipient cutaneous neoplasms. This article summarizes the effects of granulocyte-macrophage/colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta), and interferon-gamma (IFN gamma) on the modulation of antigen presentation by epidermal APC. Our data indicate that these cytokines significantly and differentially modify the ability of epidermal cells to present tumor-associated antigens and that their effects differ with regard to induction of primary immunity (sensitization) or elicitation of secondary immune responses.


Deleterious effects of cis-urocanic acid and UVB radiation on Langerhans cells and on induction of contact hypersensitivity are mediated by tumor necrosis factor-alpha. Ultraviolet B (UVB) light disrupts epidermal Langerhans cells (LC) universally and impairs the induction of contact hypersensitivity (CH) to epicutaneously applied haptens in certain strains of mice. Similar effects are observed when tumor necrosis factor-alpha (TNF alpha) is injected intradermally (ID) in mice. Trans-urocanic acid (UCA), a photoreceptor for UVB radiation, is known to be immunosuppressive. To determine whether cis-UCA is important in the process by which UVB and/or TNF alpha act in the skin, cis-UCA was injected ID into C57BL/6, C3H/HeN, BALB/c, and C3H/HeJ mice. Whole mounts of epidermis were removed 5 h later and stained immunochemically with anti-Ia antibodies. Microscopy revealed that Ia-bearing LC had lost their dendrites, had rounded up, and were reduced in number in all strains examined. Moreover, when dinitrofluorobenzene (DNFB) was applied epicutaneously to the injected site, induction of CH was grossly impaired. When neutralizing anti-TNF alpha antibodies were administered intraperitoneally 2 h prior to ID injection of cis-UCA, the deleterious effects on LC and CH induction were largely reversed. These results indicate that the actions of cis-UCA on LC and on CH induction are very similar to those achieved by ID injections of TNF alpha and by cutaneous exposure to low-dose UVB. Because the effects of UVB radiation and cis-UCA are reversed by anti-TNF alpha antibodies, we propose that UVB radiation impairs the induction of CH in mice by converting trans-UCA to cis-UCA within the epidermis; cis-UCA in turn causes the local release of TNF alpha, which thwarts sensitization by its ability to alter the functional program of epidermal Langerhans cells, thereby preventing the induction of CH.


IL-3 production as an in vitro marker of the genetically determined traits of UVB susceptibility and UVB resistance. The capacity of low-dose ultraviolet B (UVB) radiation to disrupt epidermal Langerhans cell function and to prevent the induction of contact hypersensitivity (CH) is genetically determined in mice and men. In mice, Tnf alpha and Lps are the genetic loci at which reside alleles that dictate susceptibility and resistance to the deleterious effect of UVB radiation. Detection of the UVB-susceptibility (UVB-S) and UVB-resistance (UVB-R) traits relies upon the in vivo end point of contact hypersensitivity, and is cumbersome, labor intensive, and time consuming. It has recently been reported that hapten-immune murine T cells can secrete interleukin-3 (IL-3) in vitro when exposed to hapten-derivatized syngeneic stimulator cells. To determine whether this assay might be useful in distinguishing UVB-R from UVB-S mice, panels of UVB-susceptible (C57BL/10, C3H/HeN) and UVB-resistant (A/J, BALB/c, C3H/HeJ) mice were sensitized epicutaneously with dinitrofluorobenzene (DNFB). When challenged in vitro 6 d later with dinitrophenyl-derivatized stimulator cells, T cells from all strains proliferated and secreted IL-3. Moreover, T cells from UVB-R mice that were sensitized through UVB-treated skin also made copious amounts of IL-3. However, T cells from UVB-S mice whose abdominal skin had been UVB irradiated prior to epicutaneous application of DNFB failed to secrete IL-3 in vitro, although the cells did proliferate. We conclude that following application of a sensitizing dose of hapten to UVB-exposed skin of UVB-S mice a) hapten-specific T cells are selectively unable to secrete IL-3 in vitro in response to hapten stimulation, and b) this inability is a reliable marker of the UVB-S trait. The IL-3 assay may prove useful in elucidating the mechanism by which UVB-exposed Langerhans cells activate regulatory T cells, and in detecting the UVB-S and UVB-R traits in humans.


Effects of the immunosuppressive dipeptide L-leucyl-L-leucine O-methyl ester on epidermal Langerhans cells. Pretreatment of newborn human foreskin epidermal cells (EC) with L-leucyl-L-leucine methyl ester (Leu-Leu-OME) was found to dramatically inhibit their ability to serve as alloantigen-presenting cells in a primary, one-way, allogeneic mixed epidermal cell-lymphocyte reaction (MECLR) without significantly affecting EC viability. The Leu-Leu-OME-induced MECLR inhibition could not be accounted for by a cytotoxic effect on epidermal Langerhans cells (LC), the class II major histocompatibility (MHC) antigen-bearing EC type that is fully responsible for alloantigen presentation in a primary allogeneic MECLR. Pretreatment with Leu-Leu-OME was found to inhibit the culture-induced increase in surface expression of HLA-DR molecules on LC. These findings suggest that in addition to its cytotoxic effect on cells containing high levels of dipeptidyl peptidase I, Leu-Leu-OME can also perturb alloantigen-presenting cells that are not killed by this agent by modulating HLA-D region antigen expression, perhaps through its capacity to neutralize acid compartments within cells. In addition, these studies support the notion that the culture-induced upregulation of class II MHC antigens on epidermal LC is functionally associated with the enhanced alloantigen-presenting capabilities that these cells display after short-term culture and suggest that newly synthesized class II antigens might be critical to the alloantigen-presenting cell capabilities of LC.


Accessory function of human Langerhans cells in the primary allogeneic T-cell response. Beyond antigen processing and presentation, antigen-presenting cells must convey additional signals that are necessary for T-cell activation and proliferation. In this study we analyzed the accessory function of human Langerhans cells (LC) in the primary T-cell response to alloantigens. We used paraformaldehyde-fixed LC suspensions to dissociate membrane-associated accessory structures from soluble activating factors. When freshly prepared LC-enriched (eLC, 8-20% LC) epidermal cell suspensions were fixed, normal allogeneic T cells failed to proliferate. This defect cannot be overcome by the addition of either interleukin (IL)-1 and/or IL-6 during the mixed skin cell lymphocyte reaction (MSLR). By contrast, when eLC were first incubated for 3 d, and especially in the presence of interferon gamma (IFN-gamma) before fixation, they retained significant allostimulatory property that did not correlate with increased MHC class II antigen expression. Furthermore, we showed that once LC have been activated, IL-1, but not IL-6, could serve as a co-stimulatory factor in the primary allogeneic T-cell response. In conclusion, the data suggest that human LC accessory function is not constitutive but requires an activation step, which can be provided by IFN-gamma during LC-T-cell interaction.


Infection of accessory dendritic cells by human immunodeficiency virus type 1. Many details of the pathogenesis of the human immunodeficiency virus type 1 remain to be elucidated. Details of how the virus gains entry via the mucosal surface upon sexual contact or during breast feeding remain obscure. The means by which the infection travels throughout the body as well as the nature of the major reservoirs of virus infection remains, for the most part, unknown. Recent studies raise the possibility that cells of the Langerhans/dendritic lineage play a central role in human immunodeficiency virus (HIV-1) infection and pathogenesis. It has been known for several years that veiled dendritic cells in the circulation as well as skin Langerhans are infected in people with prolonged HIV-1 infections. More recently it has been found that a large burden of viral DNA sequences is found, not only in the circulating T-cell population, but also in a population that is defined as a non-T, non-B, non-monocyte/macrophage population rich in T-helper dendritic cells. Detailed analysis of infection of primary blood-derived T-helper dendritic cells by HIV-1 shows that such cells are the most susceptible cells in the blood to infection by this virus. The cells also produce much more virus per cell than do purified populations of other blood mononuclear cells. Moreover, primary blood-derived T-helper dendritic cells are not killed by infection by HIV-1. These cells are susceptible to lymphotropic, monocyte tropic, and primary isolates of HIV-1. The sensitivity of primary blood-derived T-helper dendritic cells to infection by HIV-1 has been shown to be attributable to rapid uptake of virus particles as well as rapid synthesis of viral DNA. Subsequent steps of virus replication also occur more rapidly and more efficiently in populations of primary blood-derived T-helper dendritic cells than they do in purified preparations of blood-derived T cells and monocyte/macrophages. Studies with primates using the simian immunodeficiency virus (SIV) show that dendritic cells at the surface of sexual mucosa are rapidly infected upon exposure to high concentrations of the virus. SIV is also produced in abundance in Langerhans cells located at the surface of the sexual mucosa in animals infected for prolonged periods of time.


In vitro HIV-1 entry and replication in Langerhans cells may clarify the HIV-1 genome detection by PCR in epidermis of seropositive patients. Being dendritic antigen-presenting cells in skin and mucous membrane, Langerhans cells (LC) occur in areas at risk for inoculation by human immunodeficiency virus (HIV), and the question whether LC act as a target, reservoir, or vector for transmission of HIV has given rise to much controversy. To address this question, we first analyzed the epidermal compartment of skin from patients seropositive for HIV DNA. Second, we tested the susceptibility of each cell type normally found in this compartment to in vitro infection by HIV-1. A non-denatured DNA was obtained from epidermal sheets after a thermochemical treatment of biopsies (0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.5 at 60 degrees C for 90 seconds). Optimization of amplification of viral genome was performed with three primer pairs derived from gag, env, and pol sequences. Polymerase chain reaction (PCR) products were analyzed by Southern blot. Viral genome was found in five of 11 HIV-seropositive patients. To control the permissivity of epidermal cell population for HIV, cells isolated from the epidermal sheet of normal skin by trypsinization were co-cultured with HIV-1-carrying promonocytic cells (U937) and observed by electron microscopy. After 3-6 h of co-culture, numerous virions were either tightly bound or apparently engaged in the process of internalization through receptor-mediated endocytosis. At day 4 of co-culture, some infected LC appeared to release mature viral particles through bud formation. The in vitro HIV-1 entry and replication in LC may confirm the presence of the HIV-1 genome by PCR in epidermis of seropositive patients. The consequences of the permissivity of LC for HIV on the antigen-presenting function remain to be determined.


Regulation of complement activity by vaccinia virus complement-control protein. A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.


Differential antibody responses to rubella virus infection in males and females. Specificities of human rubella virus (RV)-specific IgG, IgM, and IgA antibodies for RV structural proteins (envelope E1 and E2 and capsid) and an E1 domain represented by a synthetic peptide (BCH-178) were determined by immunoblot and ELISA techniques in sera from rubella-infected individuals. Sequential sera were obtained from 67 females and 32 males during acute and convalescent infection phases. Males and females had differences in all antibody classes, especially during the early acute infection phase. At no time during follow-up were IgA anti-E2 antibodies detected in males, in contrast to the case in females. Significantly lower levels of IgG antibody directed to E2 were also observed in males. Males had earlier onset of E1-specific IgG and IgM antibodies with a greater proportion of total RV antibody response directed to E1 (IgG) or E1 peptide (IgM). These differences suggest there are hormonal and genetic influences on immune recognition of RV proteins that may be related to the increased incidence of rubella-associated arthropathy in females.


The role of double-stranded RNA in induction of the acute-phase response in an abortive influenza virus infection model. The inducer of the acute-phase response in "flu-like" viral infections is not defined. The hypothesis that virus-associated double-stranded (ds) RNA serves this function was investigated by comparison of several acute-phase responses (fever and sleep patterns, white and nucleated red blood cell levels, serum antiviral activity and ceruloplasmin) induced by the synthetic dsRNA polyriboinosinic:polyribocytidylic acid (poly[rI:rC]) with those induced by influenza virus in rabbits. The capacity of either dsRNA or influenza virus to induce hyporesponsiveness with respect to these acute-phase parameters upon rechallenge with the same agent or cross-challenge 24 h later was also examined. Poly(rI:rC) induced only minimal hyporesponsiveness to itself but was a potent inducer of hyporesponsiveness to virus with respect to fever, sleep, leukograms, and antiviral activity. Therefore, poly(rI:rC) can substitute for virus in terms of induction of acute-phase hyporesponsiveness, suggesting that dsRNA of viral origin triggers the acute-phase response in this model of influenza.


Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United States, 1975-1988. Cases of adult botulism (n = 309) were studied to identify clinical differences between toxin types and to evaluate the sensitivity of diagnostic laboratory testing. Patients with illness from type E toxin had the shortest incubation periods. Sporadic case-patients were more severely ill: 85% required intubation compared with only 42% in multiperson outbreaks. Of patients with type A botulism, 67% required intubation compared with 52% with type B and 39% with type E. Toxin testing was positive for 40%-44% of serum and stool specimens obtained within 3 days of toxin ingestion and for 15%-23% of specimens obtained thereafter, while 37% of stool specimens obtained > 3 days after toxin ingestion were positive by culture. Patients with type A botulism have more severe illness. In general, specimens obtained early are more likely to be positive by toxin assay, and stool cultures are more sensitive than toxin detection for specimens obtained later in the illness.


Systemic and mucosal antibody responses to toxin A in patients infected with Clostridium difficile. The systemic and mucosal humoral response to toxin A, the primary virulence factor of Clostridium difficile, was measured in sera and intestinal secretions from 21 patients with C. difficile diarrhea, 9 asymptomatic C. difficile fecal excretors, and 10 noncolonized control subjects. Toxin A-specific IgG was higher in convalescent sera of the patients with diarrhea (mean +/- SE, 990 +/- 260 ng/mL) than in acute sera (620 +/- 150 ng/mL), in sera from asymptomatic excretors (410 +/- 140 ng/mL), or control subjects (320 +/- 50 ng/mL; P < .05 convalescent vs. control). The pattern of toxin A-specific serum IgA and intestinal secretory IgA levels was similar to that of serum IgG in these groups. Neutralization of toxin A was demonstrated in 5 of 14 convalescent sera but only 1 of 13 acute sera (P = .04). However, the presence of neutralizing activity was independent of the subsequent clinical response. Therefore, most patients convalescent from C. difficile diarrhea demonstrate systemic and mucosal antibodies to toxin A, but these antibodies following natural infection do not appear to alter the clinical course of C. difficile infection.


Growth of Escherichia coli in human urine: role of salt tolerance and accumulation of glycine betaine. Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidney and excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salt-tolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains were examined. Tolerance varied from 0.1 to 0.7 M NaCl (median, 0.5) in minimal medium. There were no significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.


Antibodies against IgA1 protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis. IgA1 protease was purified from a strain of serogroup A Neisseria meningitidis subgroup IV-1, representative of bacteria that caused an epidemic of meningococcal meningitis in The Gambia in 1982-1983. ELISAs and immunoblot assays were done using this protease as antigen with paired acute- and convalescent-phase sera from patients from that epidemic and from one in Finland caused by other serogroup A meningococci. Paired sera were also tested from healthy Gambians who were persistent nasopharyngeal carriers, persistent noncarriers, or persons who became carriers after the first serum sample was taken. The results correlated well between the two methods: Antibodies were stimulated by disease or acquisition of carriage, and they remained at a constant level upon continued carriage.


Characterization of the serum antibody response to the capsular polysaccharide of Haemophilus influenzae type b in children with invasive infections. The serum antibody response to the capsular polysaccharide of Haemophilus influenzae type b (Hib) was studied in 30 children aged 1 day-5 years with invasive Hib infections. From each child, serum was obtained 0-2 days, 5-11 days, 1 month, and 6-12 months after onset of symptoms. Total antibodies were determined with RIA and isotypes with ELISA. Only 2 children had antibody levels above the estimated protective level (0.15 microgram/mL) in the first serum sample. The antibody response was age dependent with wide individual variations. Children > or = 2 years had increases in IgG, IgM, and IgA antibodies with predominance of IgG. The initial IgG response was IgG1 and IgG2 with predominance of IgG1. In the last serum sample, IgG1 antibodies had decreased while IgG2 antibodies remained unchanged. Only 2 of 7 children < 1 year had a detectable antibody response. The correlation coefficient for total antibodies compared with the sum of IgG, IgM, and IgA was .88 (P < .0001) and for IgG compared with the sum of IgG1 and IgG2 was .97 (P < .0001).


Changes in the distribution of Haemophilus influenzae type b clones associated with widespread infant vaccination in Finland. Isolates from 646 consecutive Finnish Haemophilus influenzae type b (Hib) patients with systemic disease, collected before and during large-scale vaccinations with Hib conjugate vaccines, were analyzed by major outer membrane protein (OMP) subtyping, lipopolysaccharide (LPS) serotyping, and biotyping (BT). Strains with OMP-BT-LPS combinations (clones) 1-I-1 and 1c-I-1 disappeared at the same rate as the disease they were associated with. A preferential decrease in the number of isolates of clone 1-II-1 was recorded, whereas the reduction in disease caused by strains of clone 1-II-9 occurred at a lower rate than expected. The latter clone occurred mainly in the most densely populated area of Finland. Strains belonging to all the common Hib clones were isolated from the 16 infants who acquired Hib disease despite being (partially) vaccinated. Thus, Hib clones disappeared during mass vaccination with conjugate vaccines, although at different rates.


The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio. Streptococcus pneumoniae, type 23F, resistant to penicillin (MIC, 2 micrograms/mL) and multiple other antimicrobic agents, was isolated from middle ear fluid of a child with otitis media attending a day care center in Ohio. To determine the extent of spread of this strain, nasopharyngeal culture surveys were done, and 52 carriers were identified among 250 children attending the index day care center. No carriers were found among 121 children at two other day care centers in the same urban area. Use of prophylactic doses of antibiotics (P < .001) and frequent use of antibiotics (P < 0.001) were risk factors for nasopharyngeal carriage. Carriers were more likely to have had frequent otitis media episodes (P < .02) and otitis media not responsive to antimicrobial therapy (P < .001). Strategies to limit the spread of highly resistant pneumococcal strains should include encouraging judicious use of antimicrobic agents and reevaluating indications for prophylactic use of antimicrobic agents.


Recombinant murine interferon-gamma reversibly activates rat alveolar macrophages to kill Legionella pneumophila. The interaction of interferon (IFN)-gamma, rat alveolar macrophages, and Legionella pneumophila was studied in vitro to define the effector cell potential of alveolar macrophages against an intracellular pathogen in a model in which the efficacy of IFN-gamma could be tested in vivo. Alveolar macrophages preincubated with IFN-gamma up-regulated Ia antigen and killed 0.5-4 logs of L. pneumophila over 4 days compared with 1-2 logs of bacterial growth in untreated cells. The bactericidal effect was dose dependent, evident over a wide range of bacterial inocula, and not suppressed by hydrocortisone. Preincubation with IFN-gamma was unnecessary and insufficient, as intracellular replication was reversed by exposure to IFN-gamma up to 48 h after infection, and neutralization of IFN-gamma after infection permitted bacterial growth. IFN-gamma thus converts alveolar macrophages from target cells to effector cells in host defense against L. pneumophila and may be of therapeutic benefit in legionellosis.


Risk assessment and laboratory diagnosis of trichomoniasis in men. Control of Trichomonas vaginalis is assuming higher priority because recent studies have suggested that trichomoniasis enhances susceptibility to human immunodeficiency virus infection and the risk for delivery of low-birth weight infants. In a cross-sectional study, 50 cases were identified among 447 men attending a sexually transmitted disease clinic. As previously reported, trichomoniasis was associated with nonchlamydial nongonococcal urethritis. Other risk factors included sexual contact with an infected woman or prior treatment for trichomoniasis or nongonococcal urethritis. Urethral and first-void urine cultures were positive in 80% and 68% of positive cases, respectively. When combined, these two cultures diagnosed 49 (98%) of 50 cases. These data suggest that criteria for selection of men for culture should include presence of nonchlamydial nongonococcal urethritis, recent exposure to trichomoniasis, or a history of trichomoniasis or nongonococcal urethritis. In addition, combining urethral and urine sediment cultures may prove accurate for evaluating T. vaginalis infection.


Effectiveness of a human monoclonal anti-endotoxin antibody (HA-1A) in gram-negative sepsis: relationship to endotoxin and cytokine levels. Gram-negative sepsis is caused by endotoxin-induced release of tumor necrosis factor (TNF) and other cytokines. HA-1A is a human monoclonal antibody that binds specifically to endotoxin. HA-1A should prevent death in endotoxemic patients and reduce serum levels of TNF and interleukin-6 (IL-6). This hypothesis was tested in 82 septic patients who were randomly allocated to receive a single intravenous 100-mg dose of HA-1A or placebo. Pretreatment endotoxemia was detected in 27 patients (33%). Death occurred within 28 days of treatment in 8 (73%) of 11 placebo recipients and in 5 (31%) of 16 HA-1A recipients (P = .02). The median decrease in serum TNF level 24 h after treatment was 12 ng/L in patients given HA-1A and 0 ng/L in placebo recipients (n = 65; P = .04). For IL-6, this was 204 ng/L in patients given HA-1A and 44 ng/L in placebo recipients (n = 67; P = .4). Thus, HA-1A reduces mortality in septic patients with endotoxemia and lowers serum TNF levels.


Factors associated with invasive lung aspergillosis and the significance of positive Aspergillus culture after liver transplantation. From January 1981 to December 1990, 2180 patients underwent orthotopic liver transplantation at the University of Pittsburgh. Thirty-two patients (1.5%) were identified with invasive aspergillosis (29 lung, 2 intraabdominal, 1 meningitis). Of 29 patients with invasive lung disease, only 23 (79%) had positive culture (Aspergillus fumigatus, 20; Aspergillus flavus, 3). Forty-eight variables were analyzed and compared in 23 patients with invasive disease with positive cultures and 9 patients with colonization only. The variables associated with pulmonary invasive disease, by univariate analysis, were surgical time (P = .03), presence of laparotomies (P = .02), higher creatinine level at time of Aspergillus isolation (P = .01), and use of OKT3 (P = .02). However, in a multivariate analysis, only the last two (creatinine, OKT3) were associated with invasive lung aspergillosis. Of 4 patients with positive abdominal wound culture, 2 had local invasive aspergillosis. Therefore, positive cultures of Aspergillus organisms from respiratory secretions and wound drainage may represent invasive disease and should not be ignored.


High-dose mefloquine in the treatment of multidrug-resistant falciparum malaria. The therapeutic efficacy and toxicity of a high-dose (25 mg/kg) mefloquine regimen (M25) and the currently recommended regimen of 15 mg/kg (M15) were compared in 199 patients with acute falciparum malaria in an area with deteriorating multidrug resistance on the Thai-Burmese border. The clinical and parasitologic responses were significantly more rapid with M25. The incidence of treatment failures by day 7-9 was 7% for M15 and 1% for M25 (P = .03) and had increased to 40% and 9%, respectively, by day 28 (P < .0001). Overall failure rates were highest in children (P = .02). Parasite clearance times were a good predictor of the therapeutic response; all patients with parasitemia persisting > 5 days after treatment experienced subsequent recrudescence. Side effects were dose-related and included dizziness, anorexia, nausea, vomiting, and fatigue. Although vomiting < 1 h after treatment was more likely in young children, children overall tolerated mefloquine better than adults, and men better than women. The optimum treatment dose of mefloquine in this area is 25 mg/kg.


Immunotherapy of respiratory syncytial virus infection in cotton rats (Sigmodon fulviventer) using IgG in a small-particle aerosol. To determine whether aerosolized IgG can be used effectively in the treatment of respiratory syncytial virus (RSV) infections, cotton rats were infected intranasally with RSV and treated 3 days later with human IgG containing anti-RSV antibodies delivered in a small-particle aerosol. Pulmonary histology and virus titers were determined 24 h after IgG treatment. A single 15-min exposure to aerosolized IgG did not exacerbate pulmonary pathology and effected a 50-fold reduction in pulmonary virus titer (2.95 vs. 4.67 log10 geometric mean pfu/g for untreated controls, P < .001), which was comparable to that effected by intranasally instilled IgG (50 mg/kg) (3.24 vs. 4.67 log10 geometric mean pfu/g for controls, P < .001). A 15-min exposure to aerosolized ribavirin (20 mg/mL) was not effective in reducing pulmonary virus. This study suggests that aerosolized IgG could be useful in the treatment of RSV lower respiratory tract infections and that it compares favorably with ribavirin.


Cholera in Piura, Peru: a modern urban epidemic. In late January 1991, epidemic cholera appeared in Peru. Within 2 months, 7922 cases and 17 deaths occurred in Piura, a Peruvian city of 361,868. A hospital-based culture survey showed that 79%-86% of diarrhea cases were cholera. High vibriocidal antibody titers were detected in 34% of the asymptomatic population. A study of 50 case-patients and 100 matched controls demonstrated that cholera was associated with drinking unboiled water (odds ratio [OR], 3.9; 95% confidence interval [CI], 1.7-8.9), drinking beverages from street vendors (OR, 14.6; CI, 4.2-51.2), and eating food from street vendors (OR, 24.0; CI, 3.0-191). In a second study, patients were more likely than controls to consume beverages with ice (OR, 4.0; CI, 1.1-18.3). Ice was produced from municipal water. Municipal water samples revealed no or insufficient chlorination, and fecal coliform bacteria were detected in samples from 6 of 10 wells tested. With epidemic cholera spreading throughout Latin America, these findings emphasize the importance of safe municipal drinking water.


Outbreak of cholera associated with crab brought from an area with epidemic disease. From 31 March through 3 April 1991, 8 New Jersey residents developed severe, watery diarrhea after eating crabmeat brought back in the suitcase of a traveler to Ecuador. Stool cultures yielded toxigenic Vibrio cholerae O1, serotype Inaba, biotype El Tor from 4 persons, and vibriocidal antibody titers were > or = 1:640 in 7 persons, indicating recent infection with Vibrio cholerae O1. Eating crab was statistically associated with illness (P = .006); however, no leftover crabmeat was available for testing. All 8 patients fully recovered and no cases of secondary transmission were reported. This was the first reported incident of cholera in the continental United States associated with food transported from an area with epidemic disease. Discouraging the transport of perishable souvenir seafood may prevent further outbreaks.


Controlled study of a new five-component acellular pertussis vaccine in adults and young children. A new five-component acellular pertussis (AP) vaccine containing 10 micrograms of pertussis toxoid, 5 micrograms of filamentous hemagglutinin, 5 micrograms of combined agglutinogens 2 and 3, and 3 micrograms of pertactin was evaluated in adults and young children. AP vaccine was compared with saline placebo in 31 adults, and AP vaccine combined with diphtheria and tetanus toxoids (ADTP) was compared with whole cell DTP in 41 children, ages 16-20 months, who had received whole cell DTP during infancy. AP was mildly to moderately reactogenic in adults, with pain noted within 72 h and 5-8 days after immunization. ADTP was less reactogenic than DTP in children, with significantly decreased pain, redness, irritability, and fever and less use of acetaminophen reported. No late reactions were observed in any child. The multicomponent ADTP was immunogenic, with four-fold or greater antibody rises to at least four pertussis antibody assays in all 15 immunized adults. Pertussis-specific antibody responses in children who received ADTP and DTP were similar. The multicomponent ADTP vaccine is currently being studied in a National Institute of Allergy and Infectious Diseases-sponsored efficacy study in Sweden.


Effect of catalase on regional cerebral blood flow and brain edema during the early phase of experimental pneumococcal meningitis. Previous studies have demonstrated that the radical scavenger superoxide dismutase completely blocked the increase of regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain water content during the early phase of experimental pneumococcal meningitis in the rat. To obtain information on the nature of the reactive oxygen species involved, the effect of catalase, a hydrogen peroxide scavenger, was tested. Rats injected intracisternally with live pneumococci were either untreated or received intravenous catalase. The increase of rCBF and brain water content in infected untreated rats was significantly attenuated by catalase 6 h after intracisternal challenge. ICP increased in both infected groups, with a trend toward lower ICP with catalase treatment. Cerebrospinal fluid white blood cell counts were not significantly different between infected groups. These results and previous experiments using superoxide dismutase suggest that the increase of rCBF, ICP, and brain water content is mainly caused by superoxide or superoxide reaction products.


Association of genital infection with specific Chlamydia trachomatis serovars and race. Black race is an important risk marker for Chlamydia trachomatis genital infection. To define whether C. trachomatis serovars differ by ethnic distribution, a panel of monoclonal antibodies was used to serotype 934 urethral and 581 cervical isolates from patients attending a sexually transmitted diseases clinic over 2 years. The overall serovar distribution in cervical and urethral infections was comparable, with B class serovars predominating. Significantly higher inclusion counts were observed both in younger women and in nonblacks regardless of serovar. Serovar D was less frequent among blacks at the urethral site (P = .001), while serovar Ia was more frequent in blacks at both sites (urethral, P < .001; cervical, P = .02). These associations remained significant after adjusting for age and number of inclusion-forming units by multivariate analysis. Thus, specific serovars may be associated with particular racial groups; either behavioral or biologic factors could explain these findings.


A subpopulation of Treponema pallidum is resistant to phagocytosis: possible mechanism of persistence. While untreated syphilis infection is characterized by spontaneous resolution of early lesions, a few organisms evade the host immune response and persist for many years. Macrophages are generally recognized as the effector cell responsible for bacterial clearance, and phagocytosis is enhanced by immune serum. This study examined the susceptibility of Treponema pallidum isolated at various stages of lesion resolution to opsonization and phagocytosis by macrophages in vitro. Findings suggest that the population of organisms remaining after the majority of bacteria have been cleared in vivo is resistant to phagocytosis. This may provide a mechanism for the persistence of T. pallidum in the face of an otherwise active immune response.


Antigenicity of a protective recombinant filarial protein in human bancroftian filariasis. A 92-kDa fusion protein that encodes amino acids 1-479 of a 62-kDa Brugia malayi antigen induces resistance to microfilariae in mice. The antigenicity of this recombinant protein was explored in asymptomatic residents of Wuchereria bancrofti-endemic areas of Papua New Guinea and Egypt. Unlike sera from individuals in nonendemic areas, sera from residents of endemic areas contained IgG3 antibodies (up to dilution 1:1280) reactive with the fusion protein. There was little or no recombinant antigen-specific IgG1, IgG2, IgG4, or IgE. The mean level of IgG3 antibodies to the amino acid 1-479 construct in sera of putatively "immune" adult Papua New Guineans and children in whom microfilaremia was < 4 parasites/mL of blood was not significantly different (P < .05) from those with > 1000 parasites/mL. These data indicate that the filarial antigen corresponding to the recombinant protein is highly immunogenic in naturally infected children and adults and that the isotype and magnitude of antibody reactivity with it do not correlate with the microfilaremic status of asymptomatic persons.


External morphology of the sperm of Hyalomma marginatum (Acarina: Ixodidae). Mature sperm cells of the tick Hyalomma marginatum were studied by means of scanning electron microscopy. The cell tapering end is transformed into a hemispherical structure. The rest of the cell surface is covered by longitudinal cellular processes, at the tip of the tapering end of the sperm cell an invaginated acrosomal canal can be seen.


Transplacental transfer of schistosomal antigens and antibodies. Circulating S. mansoni soluble antigens (CSA) and antibodies (CAB) were investigated in sera of 32 S. mansoni-infected pregnant mothers, their 32 newborns and 26 non-pregnant infected women. CSA levels in pregnant mothers and their newborns were significantly higher than in the 3rd group. Also, a significant positive correlation was detected between the CSA levels in sera from infected mothers and the cord blood of their newborns proving the transplacental transfer of these antigens. On the other hand, CAB levels were significantly higher in the 3rd group than in the mothers and their newborns and this could be explained by the immune complexes formation. No schistosome eggs or worms were detected in placentas and the possibility of congenital schistosomiasis was eliminated.


Isospora solimanae sp. nov. from the Egyptian hoopoe (Upupa epops major). Isospora solimanae (sp. nov.) is recorded for the first time infecting the Egyptian hoopoe (Upupa epops major). Unsporulated as well as sporulated oocysts were studied, photographed and compared with other isosporan infections of related birds. Site of infection and invasion limits were determined among naturally infected hoopoes and represented diagramatically. The rate of infection was congruent to 37.5%, and its severity considered to be non-pathogenic, since no remarkable lesions were observed. No extra-intestinal site of parasite development was recorded.


Estimation of Toxoplasma gondii antibodies in patients with cardiomyopathy. The possible involvement of Toxoplasma gondii in the pathogenesis or as a complicating factor in patients with cardiomyopathy has been investigated by the serological detection of specific antibodies by IFAT. Fifty six serum samples were collected from patients who presented with dyspnoea on effort, pericardial pains, palpitations and oedema of lower limbs. They were investigated by Echo, ECG and epidemiological history, and diagnosed as dilated, ischaemic and restrictive cardiomyopathies and myocarditis. Forty serum samples from patients with valvular lesions were examined as a control group. Twelve out of 56 samples were positive at end titre of 1:16 (21.4%). Sera with positive titres were found to occur mostly in patients with ischaemic cardiomyopathy. All samples of the control group were negative.


Cutaneous leishmaniasis as a possible predisposing factor for skin malignancy. This paper reports a 13 year old Yemenian boy with cutaneous leishmaniasis (CL.). The histopathological picture of the CL lesions showed marked dysplasia indicating premalignancy. The possibility that CL as one of the predisposing factors for skin malignancy was discussed.


Dot-ELISA as a diagnostic test in hydatid disease. Detection of circulating echinococcal antigens and antibodies were tried in 18 patients with hydatid disease and 32 healthy blood donors as controls using dot-ELISA. Hydatid cyst fluid antigen and anti-echinococcal hyperimmune rabbit sera were used for the detection of antibodies and antigens respectively. The results proved that this test is sensitive (88.9%), specific (96.9%) and rapid for detecting echinococcal antibodies. On the other hand, circulating antigens could be detected with 55.6% sensitivity. This low sensitivity was attributed to the small amounts of circulating antigens and/or immune complexes formation.


Investigation of Sarcocystis as a causative agent in cardiac disease. Sera from 56 cases with cardiomyopathy and myocarditis and 40 cases with different types of valvular diseases were tested, using the indirect fluorescent antibody test (IFAT) for the detection of antibodies to Sarcocystis. None of the cases showed specific reaction where the IFAT was negative at the cut off titre 1:8 in all the cases.


An acute case of infantile visceral leishmaniasis histopathological and histochemical studies. Some histopathological and histochemical studies were carried out on the intestines of an acute case of infantile visceral leishmaniasis. Macrophages-carrying amastigotes were seen mainly in the villous tips, mucin secretion inside the mucosal cells was mild, deposits of collagen and fibrin in the lamina propria were marked and the argentaffin cells were partially lost. The whole results were discussed.


Studies on Eimeria species naturally infecting Gerbillus pyramidum (1) Exogenous stages. Exogenous stages of two new Eimerian species, naturally infecting the Egyptian gerbil Gerbillus pyramidum were described and diagnosed in the present study. (1) The subspherical type of oocysts was 20 x 17 microns (length x width) in average. These are colourless and enveloped within an even double-layered oocyst wall. Sporulated oocysts containing four sporocysts each averaged in 10 x 8 microns, and a small spherical oocyst residual body was also observed. This type is termed Eimeria pyramidi after the species name of its host. (2) The second type of oocysts was elongated in shape measured congruent to 21 x 16 microns. These are colourless and enclosed within smooth double layered oocyst wall. Sporulated oocysts of this type were lacking oocyst residual bodies. This type is termed Eimeria gerbilli after the generic name of its host. Both types of described oocysts lack micropyle and polar cap.


Assessment of left ventricular function by Doppler echocardiography in Egyptian bilharzial patients with or without pulmonary hypertension. Forty-eight Egyptian bilharzial patients were selected for this study. Forty males and eight females. Their ages ranged from 12-60 years. All had hepatosplenomegaly with or without cardiopulmonary schistosomiasis. Also a group of ten subjects was chosen as a control group. After full clinical, laboratory and radiological examinations our subjects were subjected to: right cardiac catheterization, M-mode, two-dimensional as well as Doppler echocardiography. We found that the left ventricular geometry was distorted from its circular configuration as a result of right ventricular pressure overload as measured by the eccentricity index in bilharzial pulmonary hypertension. Also there were significant impairment of all Doppler echocardiographic parameters in those patients except time to E-peak (early diastolic flow velocity) and acceleration time of aortic flow (cm/sec2).


Histopathological and histochemical studies on the sigmoidal and rectal tissues of hepatosplenic children with gastro-intestinal troubles. Hepatosplenomegaly accompanied with different intestinal troubles is more or less a marked clinical manifestations in children. The histopathological and histochemical changes were studied in biopsied materials taken from the sigmoidal and rectal tissues of 49 children. These children had schistosomiasis mansoni (26), schistosomiasis and amoebiasis (4), schistosomiasis and tuberculosis (TB.) (2), amoebiasis (4), thalassemia (6), acute myeloid leukaemia (AML) (1), mucopolysaccharidosis (1) and bacillary dysentery (5). The pathological changes were erosion ulceration hyperplasia, atrophy, crypt-abscess and fibrosis (mucosa) and oedema, congestion, cellular infiltration (Lamina propria). The chemical changes were the mucin secretion, deposition of collagen and fibrin and activity of the argentaffin cells. Not all the disease agent had the same effect, but changes were marked mainly in children with S. mansoni and/or E. histolytica.


Evaluation of different immunodiagnostic techniques for diagnosis of hydatidosis in Egypt. In this study detection of the circulating hydatid antibodies was performed by three serological tests: Immunoelectrophoresis (IEP), Enzyme linked immunosorbent assay (ELISA) and Enzyme linked immunoelectrotransfer blot (EITB) technique. The antigen which was used in these tests was Echinococcus granulosus antigen free of host (camel) antigen by immunoaffinity chromatography. Most serum samples were pre-selected from the patients on the basis of clinical syndrome or past history of possible diagnosis of echinococcosis. In addition, physical aids to diagnosis, especially sonographic and plain X-ray examination have already been carried out. The hydatidosis group was subgrouped according to the site of the cysts in different organs. The surgical removal of hydatid cysts was taken as a sure diagnosis of hydatid disease. The outcome of comparison between the three serological tests in surgically confirmed cases, showed that ELISA technique is a good screening test (88.2% sensitivity, 88.8% specificity and 88.5% diagnostic efficacy) and EITB is a good confirmatory test (100% sensitivity, 100% specificity and 100% diagnostic efficacy).


A study on experimental cryptosporidiosis. Cryptosporidium oocysts were isolated from stools of infected patients diagnosed as cryptosporidiosis. Suckling Swiss albino mice were infected orally by these oocysts in a dose of 10(6) oocysts/mouse. Mice were divided into 9 groups according to the duration of infection 2, 3, 4, 5, 6, 7, 10, 10, 15, 21 days postinfection, corresponding non infected control groups were included. Stools of infected mice were examined twice daily to determine; infection rate, prepatent period and intensity of infection. Mice were sacrificed according to the schedule and the whole intestine was isolated and processed for histopathological examination by H & E and modified Ziehl Nielsen stain, and histochemical examination for acid and alkaline phosphatase activity. The results revealed that the infection rate was 90% and prepatent period 3-5 days and the course of infection was 21 days. The maximum pathological changes were in the ileum. These changes were reversible.


Characterization of Egyptian isolates of Trichomonas vaginalis I. Isoenzyme patterns. Three T. vaginalis isolates from Egypt were compared for their isoenzyme electrophoretic patterns on cellulose acetate. The enzymes studied were: glucose-6-dehydrogenase (G6PD); malate dehydrogenase (MDH); phosphoglucomutase (PGM); glucose phosphate isomerase (GPI); malic enzyme (ME); hexokinase (HK) and lactate dehydrogenase (LDH). The three isolates shared the same isoenzyme banding patterns of MDH; GPI; HK and LDH. Two of these isolates were similar in their banding patterns of G6PD, PGM and different from those of the third isolate. The latter was similar to one of the two isolates and different from the other in the ME isoenzyme patterns.


Comparative studies on the production of invasive larvae of Steinernema feltiae Filipjev nematodes within insect host. A comparative study has been made on the production of invasive larvae of Steinernema feltiae Filipjev within the cotton leafworm Spodoptera littoralis Boisduval and house fly Musca domestica Linnaeus. The results indicated that there is a positive correlation between the reproductive rate of invasive larvae and the initial dosages, host species and host stages. The reproductive rate of invasive larvae of Steinernema feltiae in Spodoptera littoralis imagos and caterpillars was higher than in M. domestica. At the pupal stages of M. domestica, no reproduction was obtained. Pupae were refractory to parasitism presumably owing to the effective protection afforded by the puparium. S. feltiae would be better adopted to live in S. littoralis than in M. domestica.


Fine structure studies of microgametogenesis of Eimeria adenoeides (Eimeriidae, Sporozoa) infecting turkeys in Egypt. The Ultrastructure of microgametogenesis of Eimeria adenoeides was studied in the intestinal epithelium of experimentally infected turkeys' Meleagris gallopavo gallopavo. Microgamonts were recognizable by the presence of peripherally arranged nuclei and the presence of two centrioles between each nucleus and the limiting membrane of the gamont. A nuclear spindle apparatus and an intranuclear centrocone directed toward the centriole were observed. Each young microgamont was surrounded by a very narrow parasitophorous vacuole which widened during development and contained a few intravacuolar folds. Differentiation of the microgamete began when elevations of the limiting membrane appeared above the centrioles. This event was accompanied by the segregation of nuclear content into a dense osmiophilic portion and an electron-pale portion. A gradual protrusion of the dense portion of the nucleus and developing flagella into the parasitophorous vacuole was proceeded. Microgametes had an anterior perforatorium, a dense elongate nucleus, with an anteriorly positioned mitochondrion in a small groove of the nucleus. Usually two flagella could be detected per each mature microgamete.


Assessment of interleukin-1 activity of peritoneal macrophages in mice infected with Schistosoma mansoni before and after praziquantel administration. This work was devoted to clarify the changes in interleukin-1 (IL-1) activity of peritoneal macrophages in mice experimentally infected with S. mansoni before and after praziquantel administration. A significant increase in (IL-1) activity after praziquantel administration was detected. These findings clarify the role of praziquantel in improving the immune state of the infected host.


Ultrastructural features of the tegument of adult Cynodiplostomum azimi (Trematoda:Digenea) in experimentally infected albino rats. The ultrastructure of the tegument of adult Cynodiplostomum azimi and the lesions caused by the worm at this host parasite interface in albino rats are described. The tegument consists of a syncytial distal cytoplasm (approx. 2.01-3.1 microns in thickness), bounded by an outer apical plasma membrane and an inner basal trilaminated lamina. The subtegumental cells (approx. 10.34 microns in length) are connected with the distal cytoplasm by means of cytoplasmic trabeculae. The morphology of the tegument appeared very variable. Large areas were formed of irregular folds, while other areas carried finger-like or papilla-form structures. Tegumental spines appear to have a crystalline lattice structure. Three morphologically distinct types of membrane-bound inclusion bodies were described in the tegumental/perikaryal complex. The electron-lucent elongate bodies (approx. 0.23 micron in length) were the predominant first type. The second was round-ovoid bodies (average length 0.12 micron) with a central core of electron-dense material. The third was electron-dense, rod-like in shape (0.28 micron in length) and occurred occasionally. The majority of these inclusion bodies were oriented parallel to the tegumental surface. The host's mucosa at the parasite interface showed marked flattening and partial to complete loss of mucosal microvilli. Inflammatory cells, particularly eosinophils were observed at the host-parasite interface.


Study of human and parasitic factors in relation to bancroftian filariasis in Egypt. The Human Leucocytic Antigen (HLA) and the histochemical reaction of microfilaria were studied by using cytotoxicity test and microfilarial acid phosphatase activity respectively to evaluate the role of the genetic factors and the parasitic factor in the pathogenesis of filariasis. Statistical significant association was found between HLA-B7 and the susceptibility for retaining microfilariae in blood and the development of clinical manifestations of filariasis. Histochemical study reported more or less the same results obtained in Egypt in elsewhere.