Effect of Mycobacterium leprae in peripheral nerve trunk on the evolution of skin lesions.

Comparative histological studies were made of a) 41 peripheral nerve lesions and the skin in the area of supply, and b) 12 peripheral nerve lesions and concurrent but unrelated skin lesions. In the first study, small, relatively early, histologically classifiable skin lesions were found in all cases, even though there were no clinical lesions. In every case the lesion was centered on a dermal nerve. In some cases disruption of the perineurium was associated with emergence of the lesion into the dermis and a small silent local reaction. It was concluded that there was a descending spread of the disease down the neural pathway to the dermis, although it was not necessarily associated with transport of bacilli. Although the first study showed a discrepancy in the classification between skin and nerve lesions in nearly 50% of the cases (as previously reported), the second study showed no discrepancies. It is suggested that discrepancies are relatively uncommon, and that those in the first study are exceptional. The probable explanation is that microreactions in the nerve trunks had caused a shift in classification, which was not yet reflected in the immature skin lesions. In the second study, the mature skin lesions had reached immunological equilibrium. Discrepancies in classification between skin and nerve lesions, as between concurrent skin lesions, are the result of reaction. Attention is drawn to the probable role of subliminal reactions in the evolution of infections.

The cellular exudate--Mycobacterium leprae relationship and the critical reading of skin smears.

A careful reading of conventionally stained Ziehl-Neelsen skin smear preparations in leprosy provides a number of insights into the patient's situation, including his approximate position in the spectrum. This data serves as a cross-check on the primary results of the smear examination, and aids their interpretation for the purposes of diagnosis, assessment of the response to chemotherapy and the possible onset of relapse.

Rationale for the histological spectrum of tuberculosis. A basis for classification.

There is need to re-appraise the cellular response to Mycobacterium tuberculosis. Histological analysis of 54 untreated patients with established disease demonstrated a continuous spectrum of tissue responses in which six groups correlated with evidence of resistance to bacterial multiplication. A predominance of cases in the two middle groups (82%) signified an immunological equilibrium in middle grade resistant patients that is absent in related diseases such as leprosy and cutaneous leishmaniasis. The dominant feature was necrosis, which increased progressively across the spectrum. Its form varied from minimal fibrinoid change, through fine eosinophilic necrosis, to basophilic necrosis characterized by neutrophil karyorrhexis, and finally to an almost acellular lesion with many bacilli. Cytological differentiation of the granuloma was of subsidiary significance, mature epithelioid cells being found only in high resistant cases. No correlation was found for the number of lymphocytes. This classification is thought to be an accurate reflection of the immune state in relation to antigenic load. It raises a hitherto unconsidered possibility that "caseation", a loosely applied macroscopic term, may embrace immunologically distinct states. The classification of multiple lesions was consistent. Histology offers a promising basis for further immunopathological investigation.

Events surrounding the recognition of Mycobacterium leprae in nerves.

Histological examination and immunocytochemistry of Schwann cells, macrophages, and mycobacterial antigen were used to study 48 nerves of untreated patients with leprosy. None of the patients was in reaction clinically, but microreactions, involving small clusters of Schwann cells and macrophages in all cases except LL, were marked by progressive degradation of acid-fast bacilli (AFB). This was thought to be the response to the recognition of mycobacterial antigen. In the first phase, the disintegration of one or more Schwann cells caused the release of AFB, accompanied by subacute inflammation. In the second phase, as edema and cellular infiltration subsided, the necrosis of Schwann cells was replaced by granuloma formation, mycobacterial antigen being in a soluble form. Myelinated cells harbored few degraded AFB, and there was evidence that antigen-associated myelin hastened the death of Schwann cells. Only then did antigen become immunologically detectable to induce an inflammatory response whose clearance and resolution was impeded by the restraint on cellular movement due to the structure of neural tissue. These developments were sporadic but continuous. AFB and antigen released by disintegrating Schwann cells were ingested by regenerating Schwann cells and by macrophages, producing a self-perpetuating cycle which might involve either small areas or the greater part of a fascicle, and could conceivably progress to a generalized reaction.

Classification of nerves is modified by the delayed recognition of Mycobacterium leprae.

Biopsies of 42 concurrent nerve and skin lesions across the spectrum of leprosy were classified and compared histologically and bacteriologically. Observations were made as follows: a) The bacterial load was higher in nerve than in skin lesions of the same histological classification, and it was higher in nerve than in concurrent skin lesions irrespective of classification, although not at the lepromatous pole. b) There was some discrepancy between the histological classification of nerve and skin lesions in half the cases. Skin classification appeared to represent the general tissue response and, insofar as discrepancies existed, the skin classification was thought to give the better evaluation. Nerve classification was subject to minor variations of a random nature which were thought to be the outcome of local reactions due to the build up of antigen as a result of delayed recognition in an immunologically protected situation. Upgrading or downgrading ensued locally, depending on the level of antigen at the time of its detection. In such cases, the corresponding skin classification was usually BT, which occupied a critical point in the spectrum. A certain autonomy of the response between lesions of skin and nerve suggests an explanation for downgrading reactions. Although Mycobacterium leprae, alone among mycobacteria, has some sort of affinity for Schwann cells, it is the role of the nerves as protected sites which is fundamental to the course of the disease.

Immunological factors in nasal mucosa of patients with leprosy--immunoperoxidase study.

The nature of the sub epithelial zone was established. S.E. shows IgG and IgM activity in tuberculoid group. Lepromatous group did not show any IgM or IgG response. IgE activity was seen in the lepromatous region in exudate and on the surfaces of Macrophages. Lysozyme activity was seen in the mucous acini of lepromatous leprosy.

Lysozyme as a measure of cellular dynamics in the lesions of leprosy.

The levels and distribution of lysozyme-positive cells and exudate were studied in leprosy lesions through the spectrum, in untreated and treated patients, in relapse and in reactions. Altogether 124 skin biopsies were examined by the immunoperoxidase technique. Monocytes, neutrophil-polymorphs and mast cells were the most conspicuous cells seen. Lysozyme proved to be a useful means of indexing renewal of these cells in the lesions. Peak numbers of monocytes were seen in lesions of active lepromatous leprosy (LL) and of tuberculoid leprosy (TT), at poles of opposite immunological performance. In TT the stimulus for recruitment was delayed hypersensitivity (DH). A decline in DH from TT towards the middle of the spectrum, mid-borderline, was accompanied by a fall in monocyte level. Furthermore, reacting lesions due to enhanced DH also had increased numbers of monocytes. On the other hand reactions associated with immunological deterioration were similar to active lepromatous leprosy (LL) and monocyte influx was raised in response to the stimulus of free multiplication of bacilli in both cases. In TT delayed hypersensitivity acted also to promote the rapid transformation of monocytes to epithelioid and giant cells all of which were strongly positive for lysozyme. This was in contrast to much lower levels in histologically similar macrophage-epithelioid cells of BT granulomas. Lysozyme synthesis was not seen in macrophages after ingestion of M. leprae. Early foamy change was made conspicuous by lysozyme deposited in phagocytic vacuoles, but old foam cells in regressing lepromas were negative. Lysozyme bound to dead extracellular M. leprae but not to viable or intracellular organisms. Dead bacilli or immune complexes appeared to be the stimulus for neutrophil-polymorph recruitment, mainly in reactions.

Exacerbation reactions in hyperactive lepromatous leprosy.

Exacerbation reactions (ER) are acute reactions occurring locally in histoid or other highly active lepromatous lesions with an exceptionally heavy bacterial load. Clinically, they are almost silent although they may cause ulceration and the release of viable bacilli. Histologically, the influx of polymorph neutrophils and coincident macrophage degeneration mimic erythema nodosum leprosum (ENL). Later, the signs of increased permeability or necrosis of small blood vessels and mast cell degranulation are differential features. The predominant immunoglobulin is IgE, and the main complement component is C1q, C3 being mostly undetectable. The reactions appear to be mediated in part by reagins (although eosinophils are not seen). Immune complexes probably form at antigen excess. Of equal importance may be the release from highly activated macrophages and neutrophils of hydrolases and proteases, which are capable of degrading connective tissue and other cell surfaces. This report is based on a histopathological and an immunocytological study of 13 exacerbation reactions in comparison with nonreacting hyperactive lesions and with ENL. The results support the view that the essential feature of histoid lesions is their hyperactivity.

C-reactive protein and apoB containing lipoproteins are associated with Mycobacterium leprae in lesions of human leprosy.

Skin biopsies from patients with leprosy across the spectrum from tuberculoid (TT) to lepromatous (LL), including histoid lepromas and erythema nodosum leprosum (ENL) reactions, were stained immunohistochemically for the presence of C-reactive protein (CRP) and the apolipoprotein, apoB. Mycobacterium leprae bacillary material comprising cell walls, cytoplasmic and soluble components was present with increasing abundance towards the lepromatous end of the spectrum and always stained positively with anti-CRP. M. leprae from armadillos did not stain with anti-human CRP indicating that the staining of M. leprae in human tissues was not due to a cross-reaction between anti-CRP and the organism itself. When CRP was present in large amounts apoB was also demonstrated in the same distribution. CRP was detected on bacilli and their degradation products within the cytoplasm of macrophages even in the absence of a raised serum CRP level in some ENL patients and also in two cases of advanced resolving lepromas. These findings demonstrate remarkable persistence of CRP in association with M. leprae in vivo, and raise intriguing questions about the possible role of CRP in relation to the handling of leprosy bacilli.

Erythema nodosum leprosum (ENL). Ultrastructure of the connective tissue response.

In ENL lesions of the type associated with severe damage to the connective tissue of the dermis, large quantities of bacterial debris were demonstrated by electron microscopy, although not by light microscopy. The debris, in an advanced stage of degeneration, was present in the phagosomes of decrepit macrophages, in the extracellular compartment and, in particular, bound to degenerate collagen and elastic where immunoglobulin, complement, and inflammatory mediators had been demonstrated previously. It is suggested that the complexing of mycobacterial antigen is a major factor in the causation of connective tissue damage, as well as other aspects of ENL. The reason why connective tissue involvement is so variable has not been explained.

The degradation of Mycobacterium leprae by a comparison of its staining properties.

The disintegration of Mycobacterium leprae is revealed by a study of its acid-fast component, its non-acid-fast cell walls using methenamine silver, and its BCG-positive cytoplasmic component. Solid bacilli stain by the three stain techniques used to identify these products, but the BCG component is demonstrated only with difficulty and appears granular. Degradation of M. leprae is fairly rapid in BT, BB, and BL, and clearance of bacillary products occurs almost simultaneously because of the destruction of the cell walls. However, clearance is slower in nerves and BCG-positive material persists. The breakdown of cell walls is slow in LL and their clearance is delayed, but BCG-positive material is cleared as fast as it leaks out. ENL appears to coincide with a more rapid breakdown and release of disintegration products from degenerate macrophages. The Mitsuda reaction appears as an epithelioid cell granuloma after complete degradation of M. leprae with residual BCG positive material at 30 days.

An immunoperoxidase study of immunological factors in high immune and low resistance granulomas in leprosy.

The epithelioid cell granuloma in high resistant tuberculoid (TT) leprosy was contrasted with the pure macrophage granuloma of anergic lepromatous leprosy (LL) by evaluating various immunological factors operating in these lesions. The immunoperoxidase technique using antisera to immunoglobulin IgG, IgM, complement C3, C3d and C1q and other products of macrophage secretion, lysozyme, plasminogen, a1 antitrypsin and C-reactive protein and of Ia antigens revealed peak levels in tissues of most of these factors in both types of granuloma. The tuberculoid response was linked to low antigenic load and Ia-like antigen and the lepromatous response was secondary to a high antigenic load in the absence of Ia antigen. Complement and other mediators were found intracellularly in both tuberculoid and lepromatous granulomas, but extracellularly only in tuberculoid lesions. This may indicate local hypersensitivity in the tuberculoid granuloma. It is suggested that the mediators in LL macrophages remain bound to lipids of mycobacterial degenerations in the phagocytic vacuole. Secretory cells were differently sited in the two types of granulomas: peripheral in epithelioid cell lesions and central around capillaries over the whole lesion in pure macrophage granulomas of LL. In tuberculoid leprosy many of the central vessels in the granuloma were obliterated. C1q was found in fibroblasts. However, the marked absence of fibrosis in any of the lesions of leprosy, except following severe reactions, casts some doubt on the link which has been postulated between epithelioid cells and fibroblasts as an explanation of fibrosis in granulomas.